Flow cytometer with adjustable positional offset sort deflection plates and methods of using the same

ABSTRACT

Aspects of the present disclosure include a particle sorter with a droplet deflector configured to apply a known offset deflection force to a droplet stream. Particle sorters according to certain embodiments include a flow cell, a light source, e.g., laser, for irradiating an interrogation point of the flow cell, a detector for detecting light from the interrogation point, a droplet generator for producing a droplet stream from fluid exiting the flow cell and a droplet deflector configured to apply a known offset deflection force to the droplet stream. In some cases, the droplet deflector comprises first and second plates configured to be offset from one another. Methods and particle sorting modules for applying a known offset deflection force are also provided.

CROSS-REFERENCE

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 63/041,597filed Jun. 19, 2020, the disclosure of which application is incorporatedherein by reference in its entirety.

INTRODUCTION

Flow-type particle sorting systems, such as sorting flow cytometers, areused to sort particles in a fluid sample based on at least one measuredcharacteristic of the particles. In a flow-type particle sorting system,particles, such as molecules, analyte-bound beads, or individual cells,in a fluid suspension are passed in a stream by a detection region inwhich a sensor detects particles contained in the stream of the type tobe sorted. The sensor, upon detecting a particle of the type to besorted, triggers a sorting mechanism that selectively isolates theparticle of interest. Sorted particles of interest are isolated intopartitions, such as, for example, sample containers, test tubes or wellsof a multi-well plate.

Particle sensing typically is carried out by passing the fluid stream bya detection region in which the particles are exposed to irradiatinglight, from one or more lasers, and the light scattering andfluorescence properties of the particles are measured. Particles orcomponents thereof can be labeled with fluorescent dyes to facilitatedetection, and a multiplicity of different particles or components maybe simultaneously detected by using spectrally distinct fluorescent dyesto label the different particles or components. Detection is carried outusing one or more photosensors to facilitate the independent measurementof the fluorescence of each distinct fluorescent dye.

To sort particles in the sample, a drop charging mechanism chargesdroplets of the flow stream containing a particle type to be sorted withan electrical charge at the break-off point of the flow stream. Dropletsare passed through an electrostatic field and are deflected based onpolarity and magnitude of charge on the droplet into one or morepartitions, such as sample collection containers. Uncharged droplets arenot deflected by the electrostatic field and are collected by areceptacle along the longitudinal axis of the flow stream.

SUMMARY

Aspects of the present disclosure include particle sorters with adroplet deflector configured to apply a known offset deflection force toa droplet stream. Particle sorters according to certain embodimentsinclude a flow cell, a light source, e.g., laser, for irradiating aninterrogation point of the flow cell, a detector for detecting lightfrom the interrogation point, a droplet generator for producing adroplet stream from fluid exiting the flow cell and a droplet deflectorconfigured to apply a known offset deflection force to the dropletstream.

Particle sorters according to certain embodiments include dropletdeflectors with first and second plates configured to be offset from oneanother. In embodiments, the first and second plates of the dropletdeflector are configured to be adjustably offset from one another. Insome embodiments, the first and second plates are configured to beadjustably offset from one another with respect to a horizontal plane.In such embodiments, the horizontal plane may be perpendicular to theaxis of the droplet stream. In some instances, the first plate comprisesan elongated section configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane. In certain instances, the elongated section of the first platecomprises a set screw configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane.

In some examples, the first and second plates are configured to beadjustably offset from each other by greater than 0 mm to 5 mm or more.In other examples, the first and second plates are configured to beadjustably offset from each other in increments determined by the threadpitch of a set screw used to adjust the offset.

In certain embodiments, the known offset deflection force is sufficientto offset a drop deposition position by 2 mm or more (for example, whensuch offset is measured at a distance of 140 mm below the lowest pointof the first deflection plate). In some instances, the known offsetdeflection force is sufficient to offset a drop deposition position byone droplet diameter or less (for example, when such offset is measuredat a distance of 140 mm below the lowest point of the first deflectionplate).

In embodiments, the particle sorter may further comprise a plurality ofpartitions configured to receive droplets deflected by the dropletdeflector. In some embodiments, the partitions comprise a collectioncontainer. In instances, the collection container is a multi-well plate.In some cases, the multi-well plate contains 1536 or fewer wells. Insome instances, the partitions comprise collection tubes. In examples,the diameter of each partition is 1.8 mm or less.

In embodiments, the first and second plates are configured to beparallel to one another. In some embodiments, the first and secondplates are configured to be adjustably rotated to face one another.

In certain embodiments, the second plate comprises an elongated sectionconfigured to allow the second plate to be adjustably offset from thefirst plate with respect to the horizontal plane. In some cases, theelongated section of the second plate comprises a set screw configuredto allow the second plate to be adjustably offset from the first platewith respect to the horizontal plane.

In embodiments, the droplet deflector may include an actuator, e.g., amotor, configured to adjust the offset between the first and secondplates. In some embodiments, the actuator, e.g., motor, is operablylinked to a feedback subsystem. In instances, the feedback subsystemcomprises a controller operably connected to the actuator, e.g., motor,and to a detector configured to detect a distance a droplet of thedroplet stream is offset. In some instances, the feedback subsystem isconfigured to iteratively adjust the offset between the first and secondplates.

In some embodiments, the first and second plates are metallic. Inexamples, the metallic plates are spaced apart by 1 mm or more. In otherexamples, the metallic plates are spaced apart by 3 mm or more. In somecases, the first and second plates are rectangular.

Methods for deflecting droplets with a known offset deflection force arealso provided. Methods according to certain embodiments includeirradiating with a light source an interrogation point of a flow cell,detecting light from the interrogation point with a detector, producinga droplet stream from fluid exiting the flow cell with a dropletgenerator, and deflecting droplets of the droplet stream with a dropletdeflector configured to apply a known offset deflection force to thedroplet stream.

Aspects of the present disclosure also include particle sorting modulesconfigured to apply a known offset droplet deflection force. Particlesorting modules according to certain embodiments include a dropletdeflector configured to apply a known offset deflection force to thedroplet stream. In some embodiments, the droplet deflector comprisesfirst and second plates configured to be offset from one another.

Embodiments of the invention solve the problem of lack of ability tomake fine adjustments to drop deposition position in the horizontalplane, closer to, or away from the stream position, which exists incurrent flow sorters. Embodiments of the invention provide forpositioning of drops for 1536 well microplates, and for positioningadjustments for small collection tubes or other containers. Embodimentsof the invention address the problem on sort collection devices that arerectangular and do not sit at exact right angles to the horizontal planeof the sort defection streams.

BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 depicts a schematic droplet deflector of a particle sorteraccording to the present disclosure.

FIG. 2 depicts an embodiment of a first plate according to the presentdisclosure where the first plate is configured to be adjustably offsetfrom a second plate (Offset from a second plate not shown).

FIG. 3 depicts a particle sorter according to an embodiment of thepresent disclosure comprising an offset between first and second platesin the “front-to-back” axis of a horizontal plane.

FIG. 4A depicts a schematic drawing of a particle sorter according tocertain embodiments.

FIG. 4B depicts a schematic drawing of a particle sorter according tocertain embodiments.

FIG. 5 depicts the effect of applying a known offset deflection force todroplets of a droplet stream in an embodiment of a particle sorteraccording to the present disclosure by showing the droplet depositionpositions when applying deflection forces with varying degrees of aknown offset.

FIG. 6 depicts a flow cytometer according to certain embodiments.

DETAILED DESCRIPTION

Aspects of the present disclosure include a particle sorter with adroplet deflector configured to apply a known offset deflection force toa droplet stream. Particle sorters according to certain embodimentsinclude a flow cell, a light source, e.g., laser, for irradiating aninterrogation point of the flow cell, a detector for detecting lightfrom the interrogation point, a droplet generator for producing adroplet stream from fluid exiting the flow cell and a droplet deflectorconfigured to apply a known offset deflection force to the dropletstream. Methods and particle sorting modules for applying a known offsetdeflection force are also provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

As summarized above, the present disclosure provides a particle sortercomprising a droplet deflector configured to apply a known offsetdeflection force to a droplet stream. In further describing embodimentsof the disclosure, particle sorters with first and second platesconfigured to be offset from one another, with adjustable plates, withpartitions configured to receive deflected droplets and configured to beadjusted by an actuator, e.g., a motor, and feedback subsystem are firstdescribed in greater detail. Next, methods for deflecting droplets witha known offset deflection force are described. Particle sorting modulesare also described.

Particle Sorters

Aspects of the present disclosure include particle sorters with adroplet deflector configured to apply a known offset deflection force toa droplet stream. In particular, particle sorters according to certainembodiments include a flow cell, a light source, e.g., laser, forirradiating an interrogation point of the flow cell, a detector fordetecting light from the interrogation point, a droplet generator forproducing a droplet stream from fluid exiting the flow cell and adroplet deflector configured to apply a known offset deflection force tothe droplet stream.

The term “deflect” is used herein in its conventional sense to refer toapplying a force which diverts droplets in a droplet stream from flowingalong their normal trajectory (i.e., in the absence of the deflectionforce) to a different trajectory.

By applying an “offset deflection force,” it is meant applying adeflection force to droplets of a droplet stream that is offset from astandard side-to-side oriented direction (i.e., in a direction that isdifferent than the direction from which the deflection force would beapplied absent the offset). That is, the deflection force may be askewwith reference to the direction of the deflection force prior toapplying the offset. For example, the orientation at which a deflectionforce is applied to droplets of a droplet stream may be offset in ahorizontal plane that is orthogonal to the longitudinal axis of thedroplet stream. As such, in the absence of offsetting the deflectionforce, the deflection force would be applied to the droplet streamexclusively in a “side-to-side” direction within a horizontal plane.Upon offsetting the deflection force, the deflection force may thencomprise both a “side-to-side” component and a “front-to-back”component. That is, if an x-y-z coordinate system were overlaid onto adroplet deflector, a standard droplet deflector would apply a deflectionforce exclusively along the x-axis of the coordinate system. Incontrast, a droplet deflector configured to apply a known offsetdeflection force would apply a deflection force with directionalcomponents in both the x-axis and the y-axis. In some cases, thedeflection force is offset by applying the deflection force afterrotating the orientation at which the deflection force is applied to thedroplet stream around the longitudinal axis of the droplet stream.

By applying a “known offset deflection force,” it is meant applying adeflection force to droplets of a droplet stream that is offset by anamount that is by design or predetermined. That is, in some cases, a“known offset deflection force” is a deflection force that is offset byan intended amount.

As described in greater detail herein, the subject particle sortersaccording to certain embodiments provide for a droplet deflectorcomprising first and second plates configured to be offset from oneanother. In other embodiments, the subject particle sorters according tocertain embodiments provide for first and second plates configured to beadjustably offset from one another. Sorting particles, such as cells, byemploying the subject particle sorters configured to apply a knownoffset deflection force to a droplet stream results in increased sortingefficiency, such that fewer particles of a sample are wasted (e.g., byinadvertently deflecting droplets containing target particles, such ascells, into unintended locations, such as a location other than theintended well of a multi-well plate) when sorting a sample. In somecases, the efficiency of sorting may be improved such that morevariations of particles or a larger number of particles corresponding toeach type of sorted particle may be collected and sorted when thesubject particle sorters and methods are employed. When used as part offlow cytometrically sorting a sample, the subject methods can improvethe yield of particle sorting.

In embodiments of particle sorters according to the present disclosure,droplets in the droplet stream may be diverted from their normaltrajectory along the longitudinal axis of the droplet stream by a knownoffset deflection force by a distance by 0.001 mm or more as measuredradially across a plane orthogonal to the longitudinal axis of thedroplet stream (such that such radial measurement reflects the knownoffset—i.e., is comprised of both x-axis and y-axis components withrespect to an overlaid x-y-z plane), such as 0.005 mm or more, such as0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, suchas 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 5mm or more, such as 10 mm or more, such as 15 mm or more, such as 20 mmor more, such as 25 mm or more, such as 30 mm or more, such as 35 mm ormore and including 50 mm or more. For example, the droplets in thedroplet stream may be diverted by a distance of from 0.001 mm to 100 mm,such as from 0.005 mm to 95 mm, such as from 0.001 mm to 90 mm, such asfrom 0.05 mm to 85 mm, such as from 0.01 mm to 80 mm, such as from 0.05mm to 75 mm, such as from 0.1 mm to 70 mm, such as from 0.5 mm to 65 mm,such as from 1 mm 60 mm, such as from 5 mm to 55 mm and including from10 mm to 50 mm.

Particle sorters according to embodiments of the present disclosure maybe configured for sorting particles in a sample, such as cells in abiological sample. In these embodiments, the droplet deflector of theparticle sorter is configured to apply a known offset deflection forcesufficient to deflect droplets into one or more sample collectioncontainers. Accordingly, the droplet deflector may be configured toapply a known offset deflection force such that droplets are deflectedinto sample collection containers that are 0.001 mm or more from thelongitudinal axis of the droplet stream, such as by 0.005 mm or more,such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm ormore, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm ormore, such as 5 mm or more, such as 10 mm or more, such as 15 mm ormore, such as 20 mm or more, such as 25 mm or more, such as 30 mm ormore, such as 35 mm or more and including 50 mm or more. For example,droplet deflectors may be configured to deflect droplets into samplecollection containers that are diverted from the longitudinal axis ofthe flow stream by a distance of from 0.001 mm to 100 mm, such as from0.005 mm to 95 mm, such as from 0.001 mm to 90 mm, such as from 0.05 mmto 85 mm, such as from 0.01 mm to 80 mm, such as from 0.05 mm to 75 mm,such as from 0.1 mm to 70 mm, such as from 0.5 mm to 65 mm, such as from1 mm 60 mm, such as from 5 mm to 55 mm and including from 10 mm to 50mm.

As described above, particle sorters according to embodiments comprisedroplet deflectors configured to deflect droplets in a flow stream byapplying a known offset deflection force to the droplet stream. In somecases, the different offsets of the present disclosure can be describedbased on the angle formed between the direction in which the deflectionforce is applied and the line representing the intersection of ahorizontal plane and a plate of a droplet deflector. Such anglesdescribing the offset deflection force may vary depending on thestructural configuration of the subject droplet deflector of theparticle sorter, as described in greater detail below, and may rangefrom 0.01° to 90°, such as from 0.05° to 85°, such as from 0.1° to 80°,such as from 0.5° to 75°, such as from 10° to 70°, such as from 15° to65°, such as from 20° to 60°, such as from 25° to 55° and including from30° to 50°. In other cases, the different known offset deflection forcesof the present disclosure can be described based on the degree to whichthe orientation of the deflection force is rotated around thelongitudinal axis of the droplet stream prior to applying the knownoffset deflection force. Such rotation angle describing the known offsetdeflection force may vary depending on the structural configuration ofthe subject droplet deflector of the particle sorter and may range from0.01° to 360°, such as from 0.05° to 355°, such as from 0.1° to 350°,such as from 0.5° to 300°, such as from 10° to 270°, such as from 15° to135°, such as from 20° to 90°, such as from 25° to 75° and includingfrom 30° to 50°.

In embodiments of the present disclosure, the droplet deflectorcomprises first and second plates configured to be offset from oneanother. By offsetting first and second plates from one another, it ismeant, in some cases, that upon offsetting the plates, the plates nolonger directly and immediately face one another with the droplet streamstill positioned midway between the two plates. In other words, the faceof the plates are no longer horizontally opposed in the Y axis plane. Insome cases, the plates have been offset by moving one plate along a“front-to-back” axis (i.e., y-axis), as described above. In some cases,the different offsets of the first and second plates of the presentdisclosure can be described based on the distance a plate is offset inthe “front-to-back” axis (i.e., y-axis). Such distance describing theoffset between the plates may vary depending on the structuralconfiguration of the subject droplet deflector of the particle sorterand may range from 0.01 mm to 10 mm or more, such as from 0.05 mm to 9.9mm, such as from 0.1 mm to 9 mm, such as from 0.5 mm to 7.5 mm, such asfrom 0.1 mm to 6 mm, such as from 1.5 mm to 5 mm, such as from 2 mm to 4mm and including from 2.5 mm to 3.5 mm. In some cases, the differentoffsets of the first and second plates of the present disclosure can bedescribed by the angle formed between the line connecting the midpointof the first plate with the midpoint of the second plate when the platesdirectly and immediately face one another (i.e., prior to offsetting thefirst and second plates) and the line formed between the midpoint of thefirst plate with the midpoint of the second plate after offsetting theplates from one another. Such angle describing the offset between theplates may vary depending on the structural configuration of the subjectdroplet deflector of the particle sorter, as described in greater detailbelow, and may range from 0.01° to 90°, such as from 0.05° to 85°, suchas from 0.1° to 80°, such as from 0.5° to 75°, such as from 10° to 70°,such as from 15° to 65°, such as from 20° to 60°, such as from 25° to55° and including from 30° to 50°.

In some cases, the first and second plates are configured to beadjustably offset from one another. By adjustably offset from oneanother, it is meant that the amount of the offset between the first andsecond plates is dynamically configurable. In such cases, the first andsecond plates may be adjusted to increase or decrease the offset betweenthe first and second plates as desired. In such cases, the offset may beadjusted repeatedly in order to iterate through a range of offsets andin so doing, empirically converge on a desired offset.

As described above, in some embodiments the first and second plates areconfigured to be adjustably offset from one another with respect to ahorizontal plane. The horizontal plane may be parallel to the plane inwhich collection containers that receive droplets reside—i.e., the planeof a multi-well plate. In some cases, the horizontal plane isperpendicular to the longitudinal axis of the droplet stream. Byperpendicular to the longitudinal axis of the droplet stream, it ismeant that the horizontal plane is orthogonal to the longitudinal axisof the droplet stream. By longitudinal axis of the droplet stream, it ismeant the axis along which droplets of the droplet stream flow when notinfluenced by a deflection force.

FIG. 1 depicts a schematic droplet deflector of a particle sorteraccording to the present disclosure. Droplet deflector 100 includes afirst plate 100 a and a second plate 100 b. The first plate 100 a andsecond plate 100 b of the droplet deflector 100 are configured to beadjustably offset from one another. In the embodiment shown, the offsetis indicated by the available offset positions of the first plate 100 aalong the dotted line 105. The first plate 100 a and second plate 100 bare configured to be adjustably offset from one another with respect toa horizontal plane 110 shown below the first plate 100 a and secondplate 100 b. The horizontal plane 110 is spanned by a “side-to-side”axis (i.e., x-axis) 120 and a “front-to-back” axis (i.e., y-axis) 130.In some embodiments, the drop deposition position of a droplet along the“side-to-side” axis 120 can be determined by, for example, the chargeapplied to the droplet and the voltage applied to the first plate 100 aand second plate 100 b. In embodiments, the drop deposition position ofa droplet along the “front-to-back” axis 130—i.e., the degree to whichthe drop deposition position is offset—can be determined by, forexample, the amount the first plate 100 a is offset along the availableoffset positions shown by the dotted line 105. In some instances,velocity may be employed to modulate the degree of deflection. Forexample, at lower stream velocities, drops spend longer periods of timewithin the deflection field of the plates thus increasing the influenceof the field and increasing the deflection. In such instances, lowervelocities may be employed to achieve greater deflection with a givenplate configuration.

In embodiments, the first plate may comprise an elongated sectionconfigured to allow the first plate to be adjustably offset from thesecond plate with respect to the horizontal plane. The elongated sectionmay be any convenient configuration of the first plate that enables thefirst plate to be adjustably offset from the second plate. Inembodiments, the elongated section may refer to a section that iselongated along the length of the available offset positions of thefirst plate. That is, the elongated section may be elongated along the“front-to-back” axis (i.e., the y-axis), as described above. In somecases, the elongated section may traverse the length of the availableoffset positions of the first plate and may limit the movement of thefirst plate such that the position and orientation of the first platecan only be adjusted along the “front-to-back” axis. In embodiments, theelongated section may be a keyed opening designed to mate with anopposing fixture of the droplet deflector. Such keyed opening may extendalong the lateral extent of the available offset positions, such thatthe first plate is offset by translating the first plate along thelength of the keyed opening.

In such embodiments, the elongated section of the first plate maycomprise a set screw configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane. The set screw may be any convenient screw and may be positionedin the first plate as needed to adjust the position of the first plateso as to offset it with respect to the second plate. In embodiments, thefirst plate may include a threaded hole through which the set screw isadded such that the end of the set screw protrudes through the threadedhole of the first plate. In some examples, the set screw may bepositioned such that rotating the set screw in the first plate causesthe first plate to be offset in either a “frontwards” or “backwards”direction along the “front-to-back” axis (i.e., the y-axis). Inexamples, the set screw may be positioned such that rotating the setscrew causes an end of the set screw to apply a force to a fixture ofthe droplet deflector such that, as a result of applying the force, thefirst plate is further offset in the “front-to-back” axis. Furthermore,both adjustable plates can have their set screws adjusted fully “in” orfully “out” such that both plates are moved forward or back, withoutoffset to fine adjust the whole electrostatic field that a drop willpass through if that is desired. The set screw may have any convenientlength, diameter and thread pitch. In some cases, the set screw may befinely threaded in order to better enable fine adjustments to the offsetposition of the first plate.

FIG. 2 depicts a first plate 200 according to the present disclosureconfigured to be adjustably offset. The first plate 200 configured to beadjustably offset according to the present disclosure is illustrated bycomparison with a standard plate 250 of a droplet deflector that is notconfigured to be adjustably offset. The first plate 200 includes anelongated section 210 configured to allow the first plate 200 to beadjustably offset from a second plate with respect to a horizontalplane, as described above. The elongated section 210 of the first plate200 also includes a set screw 220 configured to allow the first plate tobe adjustably offset from the second plate with respect to thehorizontal plane, as described above.

In embodiments of the particle sorter of the present disclosure, thefirst and second plates may be configured to be adjustably offset fromeach other by greater than 0 mm to 5 mm or more, such as from 0.01 mm to4.99 mm, such as from 0.05 mm to 4 mm, such as from 0.5 mm to 3.5 mm,such as from 1 mm to 3 mm, and including 1.5 mm to 2.5 mm. In suchembodiments, the first plate and the second plate may be configured toadjustably offset from each other in increments that are determined bythe thread pitch of a set screw used to adjust the offset between thefirst and second plates. In instances where the set screw includesfinely threaded pitch, the first plate and the second plate may becapable of adjustments that are finer than the increments of adjustmentavailable when the set screw includes a less finely threaded pitch. Asdescribed above, the first and second plates may be adjustably offsetfrom one another in different increments with respect to a horizontalplane. In some cases, the first plate may include an elongated sectionand a set screw configured to adjustably offset the first plate from thesecond plate by offset amounts and increments as described above.

FIG. 3 depicts a particle sorter according to an embodiment of thepresent disclosure comprising an offset between first and second platesin the “front-to-back” axis (i.e., y-axis) of a horizontal plane, asdescribed above. Particle sorter 300 according to an embodiment of thepresent disclosure includes a first deflection plate 300 a, whichincludes an elongated section 310 and a set screw 320 and is configuredto be adjustably offset from a second plate 300 b, which is a standarddeflection plate (i.e., it is not configured to be adjustably offsetaccording to the present disclosure). The “front-to-back” offset betweenthe first 300 a and second plates 300 b is depicted as the horizontalspace between dotted lines 330. The horizontal offset 330 between thefirst and second plates illustrates how the first plate is offset in thebackwards direction of the horizontal plane such that the resultingdeflection force is a known offset deflection force, i.e., it includes aknown offset in the “front-to-back” direction.

In some embodiments, the known offset deflection force is sufficient tooffset a drop deposition position by 2 mm or more. For example, adroplet may be offset in the “front-to-back” axis by 2 mm or more whenmeasured at a distance, such as, for example, a distance of 140 mm,below the lowest point of the first plate. That is, when measured at adistance of 140 mm below the lowest point of the first plate of thedroplet deflector, the droplet deflector may be configured to apply aknown offset deflection force sufficient to deflect a droplet of thedroplet stream by an offset of 2 mm or more in the “front-to-back” axisof a horizontal plane. As such, the resulting offset amount of thedroplet deposition position can be offset by 2 mm or more when measuredat 140 mm below the lowest point of the first plate. In otherembodiments, the known offset deflection force is sufficient to offset adrop deposition position by one droplet diameter or less. That is, whenmeasured at a distance below the lowest point of the first plate of thedroplet deflector, the droplet deflector may be configured to apply aknown offset deflection force to deflect a droplet of the droplet streamby an offset of only one droplet diameter or less in the “front-to-back”axis of a horizontal plane. As such, the resulting offset amount of thedroplet deposition position can be offset by only one droplet diameteror less when measured at a distance below the lowest point of the firstplate.

In embodiments of the particle sorter according to the presentdisclosure, the droplet deflector of the particle sorter is configuredsuch that the first and second plates are metallic. The metallic platesof the subject particle sorters may be formed from any suitable metalcapable of producing an electric field and may include but is notlimited to aluminum, brass, chromium, cobalt, copper, gold, indium,iron, lead, nickel, platinum, palladium, tin, steel (e.g., stainlesssteel), silver, zinc and combinations and alloys thereof, such as forexample an aluminum alloy, aluminum-lithium alloy, analuminum-nickel-copper alloy, an aluminum-copper alloy, analuminum-magnesium alloy, an aluminum-magnesium oxide alloy, analuminum-silicon alloy, an aluminum-magnesium-manganese-platinum alloy,a cobalt alloy, a cobalt-chromium alloy, a cobalt-tungsten alloy, acobalt-molybdenum-carbon alloy, acobalt-chromium-nickel-molybdenum-iron-tungsten alloy, a copper alloy, acopper-arsenic alloy, a copper-berrylium alloy, a copper-silver alloy, acopper-zine alloy (e.g., brass), a copper-tin alloy (e.g., bronze), acopper-nickel alloy, a copper-tungsten alloy, a copper-gold-silveralloy, a copper-nickel-iron alloy, a copper-manganese-tin alloy, acopper-aluminum-zinc-tin alloy, a copper-gold alloy, a gold alloy, agold-silver alloy, an indium alloy, an indium-tin alloy, an indium-tinoxide alloy, an iron alloy, an iron-chromium alloy (e.g., steel), aniron-chromium-nickel alloy (e.g., stainless steel), an iron-siliconalloy, an iron-chromium-molybdenum alloy, an iron-carbon alloy, aniron-boron alloy, an iron-magnesium alloy, an iron-manganese alloy, aniron molybdenum alloy, an iron-nickel alloy, an iron-phosphorus alloy,an iron-titanium alloy, an iron-vanadium alloy, a lead alloy, alead-antimony alloy, a lead-copper alloy, a lead-tin alloy, alead-tin-antimony alloy, a nickel alloy, anickel-manganese-aluminum-silicon alloy, a nickel-chromium alloy, anickel-copper alloy, a nickel, molybdenum-chromium-tungsten alloy, anickel-copper-iron-manganese alloy, a nickel-carbon alloy, anickel-chromium-iron alloy, a nickel-silicon alloy, a nickel-titaniumalloy, a silver alloy, a silver-copper alloy (e.g., sterling silver) asilver-coper-germanium alloy (e.g., Argentium sterling silver), asilver-gold alloy, a silver-copper-gold alloy, a silver-platinum alloy,a tin alloy, a tin-copper-antimony alloy, a tin-lead-copper alloy, atin-lead-antimony alloy, a titanium alloy, a titanium-vanadium-chromiumalloy, a titanium-aluminum alloy, a titanium-aluminum-vanadium alloy, azinc alloy, a zinc-copper alloy, a zinc-aluminum-magnesium-copper alloy,a zirconium alloy, a zirconium-tin alloy or a combination thereof.

In embodiments of the present disclosure, a known offset deflectionforce is applied to droplets in the droplet stream by applying a voltageto the first and second metallic plates of the droplet deflectorresulting in an electric field between the first and second metallicplates. Such electric field between the first and second platesaccelerates and diverts the trajectory of charged target droplets fromthe longitudinal axis of the droplet stream. In such embodiments, theknown offset deflection force resulting from the electric field mayaccelerate and divert the trajectory of target droplets from thelongitudinal axis of the droplet stream to one or more sample collectioncontainers. The voltage applied to the first and second plates to divertcharged droplets as described above may be 10 mV or more, such as 25 mVor more, such as 50 mV or more, such as 100 mV or more, such as 250 mVor more, such as 500 mV or more, such as 750 mV or more, such as 1000 mVor more, such as 2500 mV or more, such as 5000 mV or more, such as 10000V or more, such as 15000 V or more, such as 25000 V or more, such as50000 V or more and including 100000 V or more. In certain embodiments,the voltage applied to the first and second metallic plates is from 0.5kV to 15 kV, such as from 1 kV to 15 kV, such as from 1.5 kV to 12.5 kVand including from 2 kV to 10 kV. In certain embodiments, the voltageapplied to the first and second metallic plates is from 0.5 kV to 15 kV,such as from 1 kV to 15 kV, such as from 1.5 kV to 12.5 kV and includingfrom 2 kV to 10 kV. Depending on the voltage applied to the first andsecond metallic plates, the electric field strength between the metallicplates may vary, ranging from 0.001 V/m to 1×10⁷ V/m, such as from 0.01V/m to 5×10⁶ V/m, such as from 0.1 V/m to 1×10⁶ V/m, such as from 0.5V/m to 5×10⁵, such as from 1 V/m to 1×10⁵ V/m, such as from 5 V/m to5×10⁴ V/m, such as from 10 V/m to 1×10⁴ V/m and including from 50 V/m to5×10³ V/m, for example 1×10⁵ V/m to 2×10⁶ V/m.

The first and second metallic plates are spaced apart from each other bya distance sufficient to generate an electric field therebetween. Forexample, the first and second metallic plates may be spaced apart by0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, suchas 0.5 mm or more, such, as 1 mm or more, such as 1.5 mm or more, suchas 2 mm or more, such as 2.5 mm or more, such as 3 mm or more, such as3.5 mm or more, such as 4 mm or more, such as 4.5 mm or more, such as 5mm or more, such as 10 mm or more, such as 15 mm or more, such as 20 mmor more and including 25 mm or more. In some instances, the first andsecond metallic plates are spaced apart by a distance that ranges from0.01 mm to 50 mm, such as from 0.05 mm to 45 mm, such as from 0.1 mm to40 mm, such as from 0.5 mm to 35 mm, such as from 1 mm to 30 mm, such asfrom 1.5 mm to 25 mm, such as from 2 mm to 20 mm and including from 3 mmto 15 mm.

In some embodiments of the particle sorter, the first and second platesof the droplet deflector are configured to be parallel to one another.That is, even when the first plate and second plates are offset from oneanother, the plane of the first plate is parallel to the plane of thesecond plate. In some instances, the first and second plates areconfigured to be adjustably rotated to face one another. That is, ininstances where the offset between the first and second plates can beadjusted, the orientation of the first and second plates can also beadjusted so that the first and second plates remain parallel to eachother at various degrees of offset from one another. In some cases,either the first plate or the second plate or both plates are rotatedabout their respective longitudinal axes (in some cases parallel to thelongitudinal axis of the droplet stream) in order to be oriented asfacing each other.

In embodiments, the second plate of the droplet deflector of theparticle sorter comprises an elongated section configured to allow thesecond plate to be adjustably offset from the first plate with respectto the horizontal plane. The elongated section may be any convenientconfiguration of the second plate that enables the second plate to beadjustably offset from the first plate. In embodiments, the elongatedsection may refer to a section that is elongated along the length of theavailable offset positions of the second plate. That is, the elongatedsection may be elongated along the “front-to-back” axis, as describedabove. In some cases, the elongated section may traverse the length ofthe available offset positions of the second plate and may limit themovement of the second plate such that the position and orientation ofthe second plate can only be adjusted along the “front-to-back” axis. Inembodiments, the elongated section may be a keyed opening designed tomate with an opposing fixture of the droplet deflector of the particlesorter. Such keyed opening may extend along the lateral extent of theavailable offset positions, such that the second plate is offset bytranslating the second plate along the length of the keyed opening. Insuch embodiments, the elongated section of the second plate may comprisea set screw configured to allow the second plate to be adjustably offsetfrom the first plate with respect to the horizontal plane. The set screwmay be any convenient set screw and may be positioned in the secondplate as needed to adjust the position of the second plate so as tooffset it with respect to the first plate in the horizontal plane. Inembodiments, the second plate may include a threaded hole through whichthe set screw is added such that the end of the set screw protrudesthrough the threaded hole of the second plate. In some examples, the setscrew may be positioned such that rotating the set screw in the secondplate causes the second plate to be offset in either a “frontwards” or“backwards” direction along the “front-to-back” axis. In examples, theset screw may be positioned such that rotating the set screw causes anend of the set screw to apply a force to a stationary fixture of thedroplet deflector such that, as a result of applying the force, thesecond plate is further offset along the “front-to-back” axis. The setscrew may have any convenient length, diameter and thread pitch. In somecases, the set screw may be finely threaded in order to better enablefine adjustments to the offset position of the second plate.

In instances, the particle sorter according to the present disclosuremay be configured to further comprise an actuator, e.g., a motor, thatis configured to adjust the offset between the first and second plates.Where the actuator is a motor, the motor may be integrated into thedroplet deflector of the particle sorter in any convenient manner suchthat the motor is capable of automatically adjusting the offset betweenthe first and second plates. In some cases, the motor may be attached toa set screw directly or indirectly through, for example a gearingmechanism, so that upon rotation of the motor, the set screw is causedto rotate, thereby adjusting the offset between the first and secondplates. Any convenient displacement protocol may be employed as a motorconfigured to adjust the offset between the first and second plates. Insome cases, the motor may be configured with an actuated translationstage, leadscrew translation assembly, geared translation device. Themotor may comprise a stepper motor, servo motor, brushless electricmotor, brushed DC motor, micro-step drive motor, high resolution steppermotor, among other types of motors.

In embodiments, the actuator, e.g., motor, is operably linked to afeedback subsystem. The feedback subsystem may be any convenient systemfor automatically controlling the amount of an adjustable offset betweenthe first and second plates. In such embodiments, the feedback subsystemmay comprise a controller operably connected to the actuator, e.g.,motor, and to a detector configured to detect a distance a droplet ofthe droplet stream is offset. For example, the detector may beconfigured to detect the distance a droplet is displaced, including thedistance the droplet is offset by the particle sorter in the“front-to-back” axis of a horizontal plane, as described above. Ininstances, the detector may comprise any convenient camera system, suchas a camera, configured to capture images of the droplet depositionposition, and the controller may be any convenient controller, such as amicrocontroller or a microprocessor, configured to evaluate the offsetof a droplet based on an image received from the camera and adjust theoffset between the first and second plates as needed to refine theoffset of the droplet deposition position. That is, in some cases, thecontroller is configured by instructions stored on a memory operablyconnected to the controller, which when executed by the controller causethe controller to adjust the amount of offset between the first andsecond plates. In some examples, the feedback subsystem is configured toiteratively adjust the offset between the first and second plates. Thatis, the feedback subsystem may be configured to make several adjustmentsto the offset between the first and second plates such that the knownoffset deflection force is iteratively adjusted and, correspondingly,the offset of the droplet deposition position is iteratively adjusted.As a result, in some instances, the feedback subsystem may provideadditional accuracy with respect to achieving a specific offset of thedroplet deposition position. In such embodiments, the feedback subsystemmay further be configured to employ calibration particles, e.g., beads,added to the droplet stream in order to detect and measure dropletoffsets. Such beads may include, for example, Accudrop Beads, such as BDFACS™ Accudrop Beads.

The first and second plates of the droplet deflector of the subjectparticle sorters may be any suitable shape, such as a circle, oval,half-circle, crescent-shaped, star-shaped, square, triangle, rhomboid,pentagon, hexagon, heptagon, octagon, rectangle or other suitablepolygon. In certain embodiments, the first and second plates arerectangular.

In embodiments, the shape and size of the first plate may be the same ordifferent from the second plate. In some embodiments the shape of thefirst plate is the same as the second plate (e.g., both rectangular). Inother embodiments, the shape of the first plate is different from thesecond plate (e.g., the first plate is square, and the second plate isrectangular). In some instances, the dimensions of the first plate arethe same as the second plate. In one example, the width of the firstplate is the same as the second plate. In other instances, the length ofthe first plate is the same as the second plate. In still otherinstances, the width and length of the first plate are the same as thewidth and length of the second plate. In some examples, the dimensionsof the first plate are different from the second plate. In one example,the width of the first plate is different from the second plate. Inanother example, the length of the first plate is different from thesecond plate. In yet another example, both the width and the length ofthe first plate is different from the width and the length of the secondplate.

Depending on the shape of the first and second plates, the dimensions ofthe first and second plates may vary. In some embodiments, each of thefirst and second plates has a width that ranges from 0.5 mm to 10 mm,such as from 1 mm to 9.5 mm, such as from 1.5 mm to 9 mm, such as from 2mm to 8.5 mm, such as from 2.5 mm to 8 mm, such as from 3 mm to 7.5 mm,such as from 3.5 mm to 7 mm, such as from 4 mm to 6.5 mm and including awidth than ranges from 4.5 mm to 6 mm. In some cases, the widths of thefirst and second plates are the same, and in other cases, the widths ofthe first and second plates differ. The length of the first and secondplates also varies ranging from 10 mm to 500 mm, such as from 15 mm to450 mm, such as from 20 mm to 400 mm, such as from 25 mm to 350 mm, suchas from 30 mm to 300 mm, such as from 35 mm to 250 mm, such as from 40mm to 200 mm, such as from 45 mm to 150 mm and including from 50 mm to100 mm. In some cases, the lengths of the first and second plates arethe same, and in other cases, the lengths of the first and second platesdiffer. In certain embodiments, the first and second plates are anasymmetric polygon where a first end has a width that is smaller thanthe width of the second end. The width at each end may range from 0.01mm to 10 mm, such as from 0.05 mm to 9.5 mm, such as from 0.1 mm to 9mm, such as from 0.5 mm to 8.5 mm, such as from 1 mm to 8 mm, such asfrom 2 mm to 8 mm, such as from 2.5 mm to 7.5 mm and including from 3 mmto 6 mm. In certain embodiments, the first and second plates areasymmetric polygons having a first end having a width from 1 to 10 mmand a second end having a width from 2 to 5 mm. For example, the firstand second plates may be asymmetric polygons having a first end having a5 mm width and a second end having a 10 mm width. In embodiments, thesurface area of the first and second plates may vary as desired and mayrange from 0.25 to 15 cm², such as 0.5 to 14 cm², such as 0.75 to 13cm², such as 1 to 12 cm², such as 1.5 to 11 cm², and including 2 to 10cm².

In some embodiments, particle sorters of interest may include one ormore sort decision modules configured to generate a sorting decision fora particle, such as a cell, based on identifying the phenotype of thecell. Droplet deflectors may be configured for sorting particles, suchas cells, from a flow stream based on the sort decision generated by thesort decision module. As described above, the term “sorting” is usedherein in its conventional sense to refer to separating components(e.g., cells, non-cellular particles such as biological macromolecules)of a sample and in some instances delivering the separated components toone or more partitions, such as sample collection containers. Forexample, the subject particle sorters may be configured for sortingsamples having 2 or more components, such as 3 or more components, suchas 4 or more components, such as 5 or more components, such as 10 ormore components, such as 15 or more components and including sorting asample having 25 or more components. One or more of the samplecomponents may be separated from the sample and delivered to a samplecollection container, such as 2 or more sample components, such as 3 ormore sample components, such as 4 or more sample components, such as 5or more sample components, such as 10 or more sample components andincluding 15 or more sample components may be separated from the sampleand delivered to a sample collection container. In some cases, thephrase “sample components” refers to cells with differing cellphenotypes.

In some embodiments, particle sorting systems of interest are configuredto sort particles, such as cells, with an enclosed particle sortingmodule, such as those described in U.S. Patent Publication No.2017/0299493, filed on Mar. 28, 2017, the disclosure of which isincorporated herein by reference. In certain embodiments, particles(e.g., cells) of the sample are sorted using a sort decision modulehaving a plurality of sort decision units, such as those described inU.S. Provisional patent application Ser. No. 16/725,756, filed on Dec.23, 2019, the disclosure of which is incorporated herein by reference.

FIG. 4A is a schematic drawing of a particle sorter 400 in accordancewith one embodiment presented herein. In some embodiments, the particlesorter 400 is a cell sorter system. As shown in FIG. 4A, a dropformation transducer 402 (e.g., piezo-oscillator) is coupled to a fluidconduit 401, which can be coupled to, can include, or can be, a nozzle403. Within the fluid conduit 401, sheath fluid 404 hydrodynamicallyfocuses a sample fluid 406 comprising particles 409 into a moving fluidcolumn 408 (e.g., a stream). Within the moving fluid column 408,particles 409 (e.g., cells) are lined up in single file to cross amonitored area 411 (e.g., where laser-stream intersect, theinterrogation point), irradiated by an irradiation source 412 (e.g., alaser). Vibration of the drop formation transducer 402 causes movingfluid column 408 to break into a plurality of drops 410 (the dropletstream), some of which contain particles 409.

In operation, a detection station 414 (e.g., an event detector)identifies when a particle of interest (or cell of interest) crosses themonitored area 411. Detection station 414 feeds into a timing circuit428, which in turn feeds into a flash charge circuit 430. At a dropbreak off point, informed by a timed drop delay (at), a flash charge canbe applied to the moving fluid column 408 such that a drop of interestcarries a charge. The drop of interest can include one or more particlesor cells to be sorted. The charged drop can then be sorted by activatingdeflection plates (not shown) to deflect the drop into partitions, forexample, a vessel such as a collection tube or a multi-well or microwellsample plate where a partition or a well or a microwell can beassociated with drops of particular interest. As shown in FIG. 4A, thedrops can be collected in a drain receptacle 438.

A detection system 416 (e.g., a drop boundary detector) serves toautomatically determine the phase of a drop drive signal when a particleof interest passes the monitored area 411. An exemplary drop boundarydetector is described in U.S. Pat. No. 7,679,039, which is incorporatedherein by reference in its entirety. The detection system 416 allows theinstrument to accurately calculate the place of each detected particlein a drop. The detection system 416 can feed into an amplitude signal420 and/or phase 418 signal, which in turn feeds (via amplifier 422)into an amplitude control circuit 426 and/or frequency control circuit424. The amplitude control circuit 426 and/or frequency control circuit424, in turn, controls the drop formation transducer 402. The amplitudecontrol circuit 426 and/or frequency control circuit 424 can be includedin a control system.

In some implementations, sort electronics (e.g., the detection system416, the detection station 414 and a processor 440) can be coupled witha memory configured to store the detected events and a sort decisionbased thereon. The sort decision can be included in the event data for aparticle. In some implementations, the detection system 416 and thedetection station 414 can be implemented as a single detection unit orcommunicatively coupled such that an event measurement can be collectedby one of the detection system 416 or the detection station 414 andprovided to the non-collecting element.

FIG. 4B is a schematic drawing of a particle sorter in accordance withone embodiment presented herein. The particle sorter 400 shown in FIG.4B includes a first deflection plate 452 and a second deflection plate454 according to the present disclosure. A charge can be applied via astream-charging wire in a barb. This creates a stream of droplets 410containing particles 410 for analysis. The particles can be illuminatedwith one or more light sources (e.g., lasers) to generate light scatterand fluorescence information. The information for a particle is analyzedsuch as by sorting electronics or other detection system (not shown inFIG. 4B). The first 452 and second deflection plates 454 can beindependently controlled to attract or repel the charged droplet toguide the droplet toward a destination collection receptacle (e.g., oneof 472, 474, 476, or 478), such as a partition. As shown in FIG. 4B, thefirst 452 and second deflection plates 454 can be controlled to direct aparticle along a first path 462 toward the receptacle 474 or along asecond path 468 toward the receptacle 478. The first 452 and seconddeflection plates 454 may be offset from each other (e.g., by adjustingthe position of the first deflection plate 452 upwards or downwards outof the plane of the figure) such that they apply a known offsetdeflection force. In some cases, such known offset deflection force maybe applied in order to more accurately align deflected droplets withcollection receptacles 472, 474, 476, and 478. If the particle is not ofinterest (e.g., does not exhibit scatter or illumination informationwithin a specified sort range), deflection plates may allow the particleto continue along a flow path 464. Such uncharged droplets may pass intoa waste receptacle such as via aspirator 470.

The sorting electronics can be included to initiate collection ofmeasurements, receive fluorescence signals for particles, and determinehow to adjust the deflection plates to cause sorting of the particles.Example implementations of the embodiment shown in FIG. 4B include theBD FACSAria™ line of flow cytometers commercially provided by Becton,Dickinson and Company (Franklin Lakes, N.J.).

In embodiments, particle sorters according to the present disclosurefurther comprise droplet generators. Droplet generators may be anyconvenient device suitable for producing a droplet stream from fluidexiting the flow cell. In embodiments, the fluid exiting the flow cellis continuous and connected, and the droplet generator causes thecontinuous and connected fluid exiting the flow cell to form disjunctand discrete droplets. In some examples, the droplet generator is anoscillating transducer. For example, in some cases, the oscillatingtransducer is a piezo-oscillator. The vibration of the droplet generatorcauses fluid moving therein to break into a plurality of droplets of thedroplet stream. The amplitude and the frequency at which the dropletgenerator vibrates affects characteristics of the droplets and thedroplet stream formed by the droplet generator.

In instances, the particle sorter further comprises a plurality ofpartitions configured to receive droplets generated by the dropletgenerator and deflected by the droplet deflector. By partition, it ismeant any convenient container capable of receiving one or more dropletsof the droplet stream, such as droplets that contain a sorted particlesuch as a cell, sorted by the particle sorter and maintaining thecontents of the partition separate and isolated from other materials notsorted into the partition. Embodiments include more than one partition,such as two partitions, four partitions, 96 partitions or 1536 or morepartitions. Partitions may be any convenient size that is capable ofreceiving and maintaining droplets of the droplet stream, such asdroplets that contain a sorted particle such as a cell, isolated fromthe droplet stream. In some cases, partitions are sized to hold morethan one droplet, such as 10 droplets, 100 droplets, 1000 droplets,10000 droplets or more. In some embodiments, partitions comprise acollection container. In instances, the collection container is amulti-well plate. Wells of the multi-well plate may be any convenientshape. In some instances, the shape of the lateral cross section of thewells is circular; in other cases, it is rectangular or square. Wellsmay be any size with sufficient capacity for holding droplets, such asdroplets that contain a sorted particle such as a cell, as needed. Forexample, the volume of a well may be 0.001 mL or more, such as 0.005 mLor 0.015 mL or 0.1 mL or 2 mL or 5 mL or more. The multi-well plate mayinclude any number of wells. In instances, a multi-well plate mayinclude six or 12 or 24 or 48 or 96 or 384 or 1536 or 3456 or 9600 ormore wells. In some instances, the multi-well plate has 1536 or fewerwells. Wells of a multi-well plate may be arranged in any convenientpattern. In some instances, wells are arranged in a rectangular shapewith a length to width ratio of approximately two to three. In someinstances, multi-well plates of the present disclosure may conform toaccepted standards such as a standard established by the Society forBiomolecular Sciences with the ANSI-Standards. Multi-well plates may becomposed of any convenient material. In some cases, multi-well platesmay be composed of polypropylene, polystyrene or polycarbonate. In otherinstances, the partitions may comprise collection tubes. Wells may beany size and shape of a lateral cross section with sufficient capacityfor holding droplets, such as droplets that contain a sorted particlesuch as a cell, as needed. In some cases, each collection tube has alateral cross section shape of a circle with a diameter of 1.8 mm orless.

As described in detail above, particle sorters according to embodimentsof the present disclosure may be configured for sorting particles in asample, such as cells in a biological sample. In these embodiments, thedroplet deflector of the particle sorter is configured to apply a knownoffset deflection force sufficient to deflect particles flowing in adroplet stream into one or more sample collection containers. Panels A-Cof FIG. 5 illustrate the effect of applying a known offset deflectionforce to droplets of a droplet stream in an embodiment of a particlesorter according to the present disclosure by showing the dropletdeposition positions when applying known offset deflection forces withvarying degrees of a known offset. The effect of the known offsetdeflection force is seen with reference to the wells of a multi-wellplate configured to receive droplets deflected by the droplet deflectorof the particle sorter, the positions and orientations of which aresubstantially held constant between Panels A-C. The multi-well platesits in the horizontal plane orthogonal to the longitudinal axis of thedroplet stream, described above and includes a row of wells that extendexclusively in the “side-to-side” direction of the horizontal plane(i.e., the x-axis of an overlaid x-y-z coordinate system), as describedabove.

Panel A of FIG. 5 shows the resulting droplet deposition positions(i.e., the locations at which droplets are deposited) of a standarddeflection force, i.e., a deflection force that does not include a knownoffset (in other words, a deflection force where the known offset iszero). In Panel A, the multi-well plate 510 includes a row of wells 520that extend exclusively in the “side-to-side” axis of the multi-wellplate 510 and the horizontal plane. The droplet deposition positions 530result from applying a deflection force to droplets of the dropletstream without a known offset component (i.e., the known offset iszero). Because the known offset of the deflection force is zero, thedroplet deposition positions 530 extend exclusively in the“side-to-side” axis of the horizontal plane. Because the deflectionforce applied to these droplets does not include a known offsetcomponent, the droplet deposition positions 530 do not extend in the“front-to-back” axis (i.e., the y-axis of an overlaid x-y-z coordinatesystem), as described above.

Panel B of FIG. 5 shows the resulting droplet deposition positions of aknown offset deflection force that includes a known offset directingdroplets toward the “backwards” direction of the “front-to-back” axis(i.e., the y-axis of an overlaid x-y-z coordinate system). In Panel B,the multi-well plate 540 includes a row of wells 550 that extendexclusively in the “side-to-side” axis of the multi-well plate 540 andthe horizontal plane. The droplet deposition positions 560 resultingfrom applying a known offset deflection force to droplets of the dropletstream with a known offset component directed toward the “backwards”direction of the “front-to-back” axis of the multi-well plate are shownin Panel B. Because the known offset of the deflection force is directed“backwards,” the droplet deposition positions 560 extend both in the“side-to-side” axis and also towards the back of the “front-to-back”axis, as seen on the left side of the multi-well plate 540 in Panel B.Because the deflection force applied to droplets includes a known offsetcomponent directing some droplets toward the “backwards” direction, thedroplet deposition positions 560 extend, in part, in the “front-to-back”axis of the multi-well plate. That is, the droplet deposition positionsshown in Panel B resulting from the application of the known offsetdeflection force are seen as extending in both the x-axis as well as they-axis of an overlaid x-y-z plane.

Panel C of FIG. 5 shows the resulting droplet deposition positions of aknown offset deflection force that include a known offset directingdroplets toward the “frontwards” direction of the “front-to-back” axis(i.e., the y-axis of an overlaid x-y-z coordinate system). In Panel C,the multi-well plate 570 includes a row of wells 580 that extendexclusively in the “side-to-side” axis of the multi-well plate 570 andthe horizontal plane. The droplet deposition positions 590 result fromapplying a deflection force to droplets of the droplet stream with aknown offset component directed toward the “frontwards” direction of the“front-to-back” axis of the multi-well plate 570 in Panel C. Because thedeflection force applied to droplets includes a known offset componentdirecting some droplets toward the “frontwards” direction, the dropletdeposition positions 590 extend both in the “side-to-side” axis of thehorizontal plane and also towards the front of the “front-to-back” axis,as seen on the left side of the multi-well plate 570 in Panel C. Becausethe deflection force applied to droplets includes a known offsetcomponent directing droplets toward the “frontwards” direction, thedroplet deposition positions 590 extend, in part, in the “front-to-back”axis of the multi-well plate. That is, the droplet deposition positionsshown in Panel C resulting from the application of the known offsetdeflection force are seen as extending in both the x-axis as well as they-axis of an overlaid x-y-z plane.

Particle sorters of the present disclosure include a flow cell. Flowcells of the subject particle sorters include an interrogation point.The interrogation point of flow cells are configured to be irradiated bya light source. In embodiments, flow cells of the subject particlesorting systems may also include a flow cell nozzle having a nozzleorifice configured to flow a flow stream through the flow cell nozzle.In some cases, the subject particle sorter includes a flow cell with aflow cell nozzle having an orifice which propagates a fluidic sample toa sample interrogation point, where in some embodiments, the flow cellnozzle includes a proximal cylindrical portion defining a longitudinalaxis and a distal frustoconical portion which terminates in a flatsurface having the nozzle orifice that is transverse to the longitudinalaxis. The length of such proximal cylindrical portion (as measured alongthe longitudinal axis) may vary ranging from 1 mm to 15 mm, such as from1.5 mm to 12.5 mm, such as from 2 mm to 10 mm, such as from 3 mm to 9 mmand including from 4 mm to 8 mm. The length of such distal frustoconicalportion (as measured along the longitudinal axis) may also vary, rangingfrom 1 mm to 10 mm, such as from 2 mm to 9 mm, such as from 3 mm to 8 mmand including from 4 mm to 7 mm. The diameter of the chamber of the flowcell nozzle may vary, in some embodiments, ranging from 1 mm to 10 mm,such as from 2 mm to 9 mm, such as from 3 mm to 8 mm and including from4 mm to 7 mm.

In certain instances, the nozzle chamber does not include a cylindricalportion and the entire flow cell nozzle chamber is frustoconicallyshaped. In these embodiments, the length of the frustoconical nozzlechamber (as measured along the longitudinal axis transverse to thenozzle orifice), may range from 1 mm to 15 mm, such as from 1.5 mm to12.5 mm, such as from 2 mm to 10 mm, such as from 3 mm to 9 mm andincluding from 4 mm to 8 mm. The diameter of the proximal portion of thefrustoconical nozzle chamber may range from 1 mm to 10 mm, such as from2 mm to 9 mm, such as from 3 mm to 8 mm and including from 4 mm to 7 mm.

In embodiments, a sample flow stream may emanate from an orifice at thedistal end of the flow cell nozzle of the flow cell. Depending on thedesired characteristics of such flow stream, the flow cell nozzleorifice may be any suitable shape where cross-sectional shapes ofinterest include, but are not limited to: rectilinear cross sectionalshapes, e.g., squares, rectangles, trapezoids, triangles, hexagons,etc., curvilinear cross-sectional shapes, e.g., circles, ovals, as wellas irregular shapes, e.g., a parabolic bottom portion coupled to aplanar top portion. In certain embodiments, flow cell nozzle of interesthas a circular orifice. The size of the nozzle orifice may vary, in someembodiments ranging from 1 μm to 20,000 μm, such as from 2 μm to 17,500μm, such as from 5 μm to 15,000 μm, such as from 10 μm to 12,500 μm,such as from 15 μm to 10,000 μm, such as from 25 μm to 7,500 μm, such asfrom 50 μm to 5,000 μm, such as from 75 μm to 1,000 μm, such as from 100μm to 750 μm and including from 150 μm to 500 μm. In certainembodiments, the nozzle orifice is 100 μm.

In some embodiments, the flow cell nozzle of the flow cell includes asample injection port configured to provide a sample to the flow cellnozzle. In embodiments, the sample injection system is configured toprovide suitable flow of sample to the flow cell nozzle chamber.Depending on the desired characteristics of the flow stream, the rate ofsample conveyed to the flow cell nozzle chamber by the sample injectionport may be 1 μL/sec or more, such as 2 μL/sec or more, such as 3 μL/secor more, such as 5 μL/sec or more, such as 10 μL/sec or more, such as 15μL/sec or more, such as 25 μL/sec or more, such as 50 μL/sec or more,such as 100 μL/sec or more, such as 150 μL/sec or more, such as 200μL/sec or more, such as 250 μL/sec or more, such as 300 μL/sec or more,such as 350 μL/sec or more, such as 400 μL/sec or more, such as 450μL/sec or more and including 500 μL/sec or more. For example, the sampleflow rate may range from 1 μL/sec to about 500 μL/sec, such as from 2μL/sec to about 450 μL/sec, such as from 3 μL/sec to about 400 μL/sec,such as from 4 μL/sec to about 350 μL/sec, such as from 5 μL/sec toabout 300 μL/sec, such as from 6 μL/sec to about 250 μL/sec, such asfrom 7 μL/sec to about 200 μL/sec, such as from 8 μL/sec to about 150μL/sec, such as from 9 μL/sec to about 125 μL/sec and including from 10μL/sec to about 100 μL/sec.

The sample injection port may be an orifice positioned in a wall of thenozzle chamber or may be a conduit positioned at the proximal end of thenozzle chamber. Where the sample injection port is an orifice positionedin a wall of the nozzle chamber, the sample injection port orifice maybe any suitable shape where cross-sectional shapes of interest include,but are not limited to: rectilinear cross sectional shapes, e.g.,squares, rectangles, trapezoids, triangles, hexagons, etc., curvilinearcross-sectional shapes, e.g., circles, ovals, etc., as well as irregularshapes, e.g., a parabolic bottom portion coupled to a planar topportion. In certain embodiments, the sample injection port has acircular orifice. The size of the sample injection port orifice may varydepending on shape, in certain instances, having an opening ranging from0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, such asfrom 0.75 mm to 2.25 mm, such as from 1 mm to 2 mm and including from1.25 mm to 1.75 mm, for example 1.5 mm.

In certain instances, the sample injection port is a conduit positionedat a proximal end of the flow cell nozzle chamber of the flow cell. Forexample, the sample injection port may be a conduit positioned to havethe orifice of the sample injection port in line with the flow cellnozzle orifice. Where the sample injection port is a conduit positionedin line with the flow cell nozzle orifice, the cross-sectional shape ofthe sample injection tube may be any suitable shape wherecross-sectional shapes of interest include, but are not limited to:rectilinear cross sectional shapes, e.g., squares, rectangles,trapezoids, triangles, hexagons, etc., curvilinear cross-sectionalshapes, e.g., circles, ovals, as well as irregular shapes, e.g., aparabolic bottom portion coupled to a planar top portion. The orifice ofthe conduit may vary depending on shape, in certain instances, having anopening ranging from 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mmto 2.5 mm, such as from 0.75 mm to 2.25 mm, such as from 1 mm to 2 mmand including from 1.25 mm to 1.75 mm, for example 1.5 mm. The shape ofthe tip of the sample injection port may be the same or different fromthe cross-section shape of the sample injection tube. For example, theorifice of the sample injection port may include a beveled tip having abevel angle ranging from 1° to 10°, such as from 2° to 9°, such as from3° to 8°, such as from 4° to 7° and including a bevel angle of 5°.

In some embodiments, the flow cell nozzle of the flow cell also includesa sheath fluid injection port configured to provide a sheath fluid tothe flow cell nozzle. In embodiments, the sheath fluid injection systemis configured to provide a flow of sheath fluid to the flow cell nozzlechamber, for example in conjunction with the sample to produce alaminated flow stream of sheath fluid surrounding the sample flowstream. Depending on the desired characteristics of the flow stream, therate of sheath fluid conveyed to the flow cell nozzle chamber by the maybe 254/sec or more, such as 50 μL/sec or more, such as 75 μL/sec ormore, such as 100 μL/sec or more, such as 250 μL/sec or more, such as500 μL/sec or more, such as 750 μL/sec or more, such as 1000 μL/sec ormore and including 2500 μL/sec or more. For example, the sheath fluidflow rate may range from 1 μL/sec to about 500 μL/sec, such as from 2μL/sec to about 450 μL/sec, such as from 3 μL/sec to about 400 μL/sec,such as from 4 μL/sec to about 350 μL/sec, such as from 5 μL/sec toabout 300 μL/sec, such as from 6 μL/sec to about 250 μL/sec, such asfrom 7 μL/sec to about 200 μL/sec, such as from 8 μL/sec to about 150μL/sec, such as from 9 μL/sec to about 125 μL/sec and including from 10μL/sec to about 100 μL/sec.

In some embodiments, the sheath fluid injection port is an orificepositioned in a wall of the nozzle chamber of the flow cell. The sheathfluid injection port orifice may be any suitable shape wherecross-sectional shapes of interest include, but are not limited to:rectilinear cross sectional shapes, e.g., squares, rectangles,trapezoids, triangles, hexagons, etc., curvilinear cross-sectionalshapes, e.g., circles, ovals, as well as irregular shapes, e.g., aparabolic bottom portion coupled to a planar top portion. The size ofthe sample injection port orifice may vary depending on shape, incertain instances, having an opening ranging from 0.1 mm to 5.0 mm,e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, such as from 0.75 mm to2.25 mm, such as from 1 mm to 2 mm and including from 1.25 mm to 1.75mm, for example 1.5 mm.

The particle sorter also includes an interrogation point (i.e., a sampleinterrogation point). In some cases, the sample interrogation point isin fluid communication with the flow cell nozzle orifice. As describedin greater detail below, a sample flow stream may emanate from anorifice at the distal end of the flow cell nozzle and particles in theflow stream may be irradiated with a light source at the sampleinterrogation point of the flow cell of the particle sorter. The size ofthe interrogation point of the particle sorter may vary depending on theproperties of the flow nozzle, such as the size of the nozzle orificeand sample injection port size. In embodiments, the interrogation pointmay have a width that is 0.01 mm or more, such as 0.05 mm or more, suchas 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as2 mm or more, such as 3 mm or more, such as 5 mm or more and including10 mm or more. The length of the interrogation point may also vary,ranging in some instances along 0.01 mm or more, such as 0.1 mm or more,such as 0.5 mm or more, such as 1 mm or more, such as 1.5 mm or more,such as 2 mm or more, such as 3 mm or more, such as 5 mm or more, suchas 10 or more, such as 15 mm or more, such as 20 mm or more, such as 25mm or more and including 50 mm or more of the particle sorter.

The interrogation point of the flow cell of the particle sorter may beconfigured to facilitate irradiation of a planar cross-section of anemanating flow stream or may be configured to facilitate irradiation ofa diffuse field (e.g., with a diffuse laser or lamp) of a predeterminedlength. In some embodiments, the interrogation point of the flow cellincludes a transparent window that facilitates irradiation of apredetermined length of an emanating flow stream, such as 1 mm or more,such as 2 mm or more, such as 3 mm or more, such as 4 mm or more, suchas 5 mm or more and including 10 mm or more. Depending on the lightsource used to irradiate the emanating flow stream (as described below),the interrogation region of the particle sorting module may beconfigured to pass light that ranges from 100 nm to 1500 nm, such asfrom 150 nm to 1400 nm, such as from 200 nm to 1300 nm, such as from 250nm to 1200 nm, such as from 300 nm to 1100 nm, such as from 350 nm to1000 nm, such as from 400 nm to 900 nm and including from 500 nm to 800nm. As such, the flow cell of the particle sorter at the interrogationpoint may be formed from any transparent material which passes thedesired range of wavelength, including but not limited to optical glass,borosilicate glass, Pyrex glass, ultraviolet quartz, infrared quartz,sapphire as well as plastic, such as polycarbonates, polyvinyl chloride(PVC), polyurethanes, polyethers, polyamides, polyimides, or copolymersof these thermoplastics, such as PETG (glycol-modified polyethyleneterephthalate), among other polymeric plastic materials, includingpolyester, where polyesters of interest may include, but are not limitedto poly(alkylene terephthalates) such as poly(ethylene terephthalate)(PET), bottle-grade PET (a copolymer made based on monoethylene glycol,terephthalic acid, and other comonomers such as isophthalic acid,cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT), andpoly(hexamethylene terephthalate); poly(alkylene adipates) such aspoly(ethylene adipate), poly(1,4-butylene adipate), andpoly(hexamethylene adipate); poly(alkylene suberates) such aspoly(ethylene suberate); poly(alkylene sebacates) such as poly(ethylenesebacate); poly(c-caprolactone) and poly(β-propiolactone); poly(alkyleneisophthalates) such as poly(ethylene isophthalate); poly(alkylene2,6-naphthalene-dicarboxylates) such as poly(ethylene2,6-naphthalene-dicarboxylate); poly(alkylene sulfonyl-4,4′-dibenzoates)such as poly(ethylene sulfonyl-4,4′-dibenzoate); poly(p-phenylenealkylene dicarboxylates) such as poly(p-phenylene ethylenedicarboxylates); poly(trans-1,4-cyclohexanediylalkylene dicarboxylates)such as poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate);poly(1,4-cyclohexane-dimethylene alkylene dicarboxylates) such aspoly(1,4-cyclohexane-dimethylene ethylene dicarboxylate);poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylene dicarboxylates) suchas poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylene dicarboxylate);lactic acid polymers and copolymers such as (S)-polylactide,(R,S)-polylactide, poly(tetramethylglycolide), andpoly(lactide-co-glycolide); and polycarbonates of bisphenol A,3,3′-dimethylbisphenol A, 3,3′,5,5′-tetrachlorobisphenol A,3,3′,5,5′-tetramethylbisphenol A; polyamides such as poly(p-phenyleneterephthalamide); polyesters, e.g., polyethylene terephthalates, e.g.,Mylar™ polyethylene terephthalate; etc. In some embodiments, flow cellsof interest include a cuvette positioned in the sample interrogationpoint. In embodiments, the cuvette may pass light that ranges from 100nm to 1500 nm, such as from 150 nm to 1400 nm, such as from 200 nm to1300 nm, such as from 250 nm to 1200 nm, such as from 300 nm to 1100 nm,such as from 350 nm to 1000 nm, such as from 400 nm to 900 nm andincluding from 500 nm to 800 nm.

In some embodiments, the sample interrogation point includes one or moreoptical adjustment components. By “optical adjustment” is meant thatlight irradiated onto the sample interrogation point or light collectedfrom an irradiated flow stream is changed as desired. In someembodiments, the sample interrogation point includes an opticaladjustment component for adjusting the light irradiated onto the sampleinterrogation point by a light source. In other embodiments, the sampleinterrogation point includes an optical adjustment component foradjusting light emanating from an irradiated flow stream before beingconveyed to a detector for measurement. In yet other embodiments, thesample interrogation point includes an optical adjustment component foradjusting both the light irradiated onto the sample interrogation pointby a light source and the light emanating from an irradiated flow streambefore being conveyed to a detector for measurement. For example, theoptical adjustment may be to increase the dimensions of the light, thefocus of the light or to collimate the light. In some instances, opticaladjustment is a magnification protocol so as to increase the dimensionsof the light (e.g., beam spot), such as increasing the dimensions by 5%or more, such as by 10% or more, such as by 25% or more, such as by 50%or more and including increasing the dimensions by 75% or more. In otherembodiments, optical adjustment includes focusing the collected thelight so as to reduce the light dimensions, such as by 5% or greater,such as by 10% or greater, such as by 25% or greater, such as by 50% orgreater and including reducing the dimensions of the beam spot by 75% orgreater. In certain embodiments, optical adjustment includes collimatingthe light. The term “collimate” is used in its conventional sense torefer to the optically adjusting the collinearity of light propagationor reducing divergence by the light of from a common axis ofpropagation. In some instances, collimating includes narrowing thespatial cross section of a light beam.

Optical adjustment components may be any convenient device or structurewhich provides the desired change in the collected light and mayinclude, but is not limited to, lenses, mirrors, pinholes, slits,gratings, light refractors, and any combinations thereof. The particlesorter may include one or more optical adjustment components at thesample interrogation point as needed, such as two or more, such as threeor more, such as four or more and including five or more opticaladjustment components.

In some embodiments, the optical adjustment component is a focusing lenshaving a magnification ratio of from 0.1 to 0.95, such as amagnification ratio of from 0.2 to 0.9, such as a magnification ratio offrom 0.3 to 0.85, such as a magnification ratio of from 0.35 to 0.8,such as a magnification ratio of from 0.5 to 0.75 and including amagnification ratio of from 0.55 to 0.7, for example a magnificationratio of 0.6. For example, the focusing lens is, in certain instances, adouble achromatic de-magnifying lens having a magnification ratio ofabout 0.6. The focal length of the focusing lens may vary, ranging from5 mm to 20 mm, such as from 6 mm to 19 mm, such as from 7 mm to 18 mm,such as from 8 mm to 17 mm, such as from 9 mm to 16 and including afocal length ranging from 10 mm to 15 mm. In certain embodiments, thefocusing lens has a focal length of about 13 mm.

In other embodiments, the optical adjustment component is a collimator.The collimator may be any convenient collimating protocol, such as oneor more mirrors or curved lenses or a combination thereof. For example,the collimator is in certain instances a single collimating lens. Inother instances, the collimator is a collimating mirror. In yet otherinstances, the collimator includes two lenses. In still other instances,the collimator includes a mirror and a lens. Where the collimatorincludes one or more lenses, the focal length of the collimating lensmay vary, ranging from 5 mm to 40 mm, such as from 6 mm to 37.5 mm, suchas from 7 mm to 35 mm, such as from 8 mm to 32.5 mm, such as from 9 mmto 30 mm, such as from 10 mm to 27.5 mm, such as from 12.5 mm to 25 mmand including a focal length ranging from 15 mm to 20 mm.

In certain embodiments, the optical adjustment component is a wavelengthseparator. The term “wavelength separator” is used herein in itsconventional sense to refer to an optical protocol for separatingpolychromatic light into its component wavelengths. Wavelengthseparation, according to certain embodiments, may include selectivelypassing or blocking specific wavelengths or wavelength ranges of thepolychromatic light. Wavelength separation protocols of interest whichmay be a part of or combined with flow cell nozzles discussed above,include but are not limited to, colored glass, bandpass filters,interference filters, dichroic mirrors, diffraction gratings,monochromators and combinations thereof, among other wavelengthseparating protocols. In some embodiments, the wavelength separator isan optical filter. For example, the optical filter may be a bandpassfilter having minimum bandwidths ranging from 2 nm to 100 nm, such asfrom 3 nm to 95 nm, such as from 5 nm to 95 nm, such as from 10 nm to 90nm, such as from 12 nm to 85 nm, such as from 15 nm to 80 nm andincluding bandpass filters having minimum bandwidths ranging from 20 nmto 50 nm.

In embodiments, the light source of the particle sorter may be anysuitable broadband or narrow band source of light. In some cases,depending on the components of a sample at the interrogation point ofthe flow cell (e.g., cells, calibration particles such as beads,non-cellular particles, etc.), the light source may be configured toemit wavelengths of light that vary, ranging from 200 nm to 1500 nm,such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such asfrom 350 nm to 900 nm and including from 400 nm to 800 nm. For example,the light source may include a broadband light source emitting lighthaving wavelengths from 200 nm to 900 nm. In other instances, the lightsource includes a narrow band light source emitting a wavelength rangingfrom 200 nm to 900 nm. For example, the light source may be a narrowband LED (1 nm-25 nm) emitting light having a wavelength ranging between200 nm to 900 nm. In certain embodiments, the light source is a laser.In some instances, the subject particle sorters include a gas laser,such as a helium-neon laser, argon laser, krypton laser, xenon laser,nitrogen laser, CO₂ laser, CO laser, argon-fluorine (ArF) excimer laser,krypton-fluorine (KrF) excimer laser, xenon chlorine (XeCl) excimerlaser or xenon-fluorine (XeF) excimer laser or a combination thereof. Inother instances, the subject particle sorters include a dye laser, suchas a stilbene, coumarin or rhodamine laser. In yet other instances,lasers of interest include a metal-vapor laser, such as a helium-cadmium(HeCd) laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser,helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser,copper laser or gold laser and combinations thereof. In still otherinstances, the subject particle sorters include a solid-state laser,such as a ruby laser, an Nd:YAG laser, NdCrYAG laser, Er:YAG laser,Nd:YLF laser, Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser,titanium sapphire laser, thulim YAG laser, ytterbium YAG laser,ytterbium₂O₃ laser or cerium doped lasers and combinations thereof.

In other embodiments, the light source is a non-laser light source, suchas a lamp, including but not limited to a halogen lamp, deuterium arclamp, xenon arc lamp, a light-emitting diode, such as a broadband LEDwith continuous spectrum, superluminescent emitting diode, semiconductorlight emitting diode, wide spectrum LED white light source, a multi-LEDintegrated light source. In some instances, the non-laser light sourceis a stabilized fiber-coupled broadband light source, white lightsource, among other light sources or any combination thereof.

The light source may be positioned any suitable distance from a sampleat the interrogation point of the flow cell (e.g., a flow stream in aflow cytometer), such as at a distance of 0.001 mm or more from the flowstream, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mmor more, such as 5 mm or more, such as 10 mm or more, such as 25 mm ormore and including at a distance of 100 mm or more. In addition, thelight source may irradiate the sample at the interrogation point of theflow cell at any suitable angle (e.g., relative the vertical axis of theflow stream), such as at an angle ranging from 10° to 90°, such as from15° to 85°, such as from 20° to 80°, such as from 25° to 75° andincluding from 30° to 60°, for example at a 90° angle.

The light source may be configured to irradiate a sample at theinterrogation point of the flow cell continuously or in discreteintervals. In some instances, particle sorters include a light sourcethat is configured to irradiate a sample continuously, such as with acontinuous wave laser that continuously irradiates, e.g., a flow streamat the interrogation point in a flow cytometer. In other instances,particle sorters of interest include a light source that is configuredto irradiate a sample at discrete intervals, such as every 0.001milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1millisecond, every 10 milliseconds, every 100 milliseconds and includingevery 1000 milliseconds, or some other interval. Where the light sourceis configured to irradiate the sample at discrete intervals, particlesorters may include one or more additional components to provide forintermittent irradiation of a sample with the light source. For example,the subject particle sorters in these embodiments may include one ormore laser beam choppers, manually or computer controlled beam stops forblocking and exposing the sample to the light source.

In some embodiments, the light source is a laser. Lasers of interest mayinclude pulsed lasers or continuous wave lasers. For example, the lasermay be a gas laser, such as a helium-neon laser, argon laser, kryptonlaser, xenon laser, nitrogen laser, CO₂ laser, CO laser, argon-fluorine(ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenonchlorine (XeCl) excimer laser or xenon-fluorine (XeF) excimer laser or acombination thereof; a dye laser, such as a stilbene, coumarin orrhodamine laser; a metal-vapor laser, such as a helium-cadmium (HeCd)laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser,helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser,copper laser or gold laser and combinations thereof; a solid-statelaser, such as a ruby laser, an Nd:YAG laser, NdCrYAG laser, Er:YAGlaser, Nd:YLF laser, Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser,titanium sapphire laser, thulim YAG laser, ytterbium YAG laser,ytterbium₂O₃ laser or cerium doped lasers and combinations thereof; asemiconductor diode laser, optically pumped semiconductor laser (OPSL),or a frequency doubled or frequency tripled implementation of any of theabove mentioned lasers.

In certain embodiments, the light source is a light beam generator thatis configured to generate two or more beams of frequency shifted light.In some instances, the light beam generator includes a laser, aradiofrequency generator configured to apply radiofrequency drivesignals to an acousto-optic device to generate two or more angularlydeflected laser beams. In these embodiments, the laser may be a pulsedlasers or continuous wave laser, such as described above.

The acousto-optic device may be any convenient acousto-optic protocolconfigured to frequency shift laser light using applied acoustic waves.In certain embodiments, the acousto-optic device is an acousto-opticdeflector. The acousto-optic device in the subject system is configuredto generate angularly deflected laser beams from the light from thelaser and the applied radiofrequency drive signals. The radiofrequencydrive signals may be applied to the acousto-optic device with anysuitable radiofrequency drive signal source, such as a direct digitalsynthesizer (DDS), arbitrary waveform generator (AWG), or electricalpulse generator.

In embodiments, a controller is configured to apply radiofrequency drivesignals to the acousto-optic device to produce the desired number ofangularly deflected laser beams in the output laser beam, such as beingconfigured to apply 3 or more radiofrequency drive signals, such as 4 ormore radiofrequency drive signals, such as 5 or more radiofrequencydrive signals, such as 6 or more radiofrequency drive signals, such as 7or more radiofrequency drive signals, such as 8 or more radiofrequencydrive signals, such as 9 or more radiofrequency drive signals, such as10 or more radiofrequency drive signals, such as 15 or moreradiofrequency drive signals, such as 25 or more radiofrequency drivesignals, such as 50 or more radiofrequency drive signals and includingbeing configured to apply 100 or more radiofrequency drive signals.

In some instances, to produce an intensity profile of the angularlydeflected laser beams in the output laser beam, the controller isconfigured to apply radiofrequency drive signals having an amplitudethat varies such as from about 0.001 V to about 500 V, such as fromabout 0.005 V to about 400 V, such as from about 0.01 V to about 300 V,such as from about 0.05 V to about 200 V, such as from about 0.1 V toabout 100 V, such as from about 0.5 V to about 75 V, such as from about1 V to 50 V, such as from about 2 V to 40 V, such as from 3 V to about30 V and including from about 5 V to about 25 V. Each appliedradiofrequency drive signal has, in some embodiments, a frequency offrom about 0.001 MHz to about 500 MHz, such as from about 0.005 MHz toabout 400 MHz, such as from about 0.01 MHz to about 300 MHz, such asfrom about 0.05 MHz to about 200 MHz, such as from about 0.1 MHz toabout 100 MHz, such as from about 0.5 MHz to about 90 MHz, such as fromabout 1 MHz to about 75 MHz, such as from about 2 MHz to about 70 MHz,such as from about 3 MHz to about 65 MHz, such as from about 4 MHz toabout 60 MHz and including from about 5 MHz to about 50 MHz.

In certain embodiments, the controller has a processor having memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to produce an output laser beam with angularly deflectedlaser beams having a desired intensity profile. For example, the memorymay include instructions to produce two or more angularly deflectedlaser beams with the same intensities, such as 3 or more, such as 4 ormore, such as 5 or more, such as 10 or more, such as 25 or more, such as50 or more and including memory that may include instructions to produce100 or more angularly deflected laser beams with the same intensities.In other embodiments, the memory may include instructions to produce twoor more angularly deflected laser beams with different intensities, suchas 3 or more, such as 4 or more, such as 5 or more, such as 10 or more,such as 25 or more, such as 50 or more and including memory that mayinclude instructions to produce 100 or more angularly deflected laserbeams with different intensities.

In certain embodiments, the controller has a processor having memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to produce an output laser beam having increasingintensity from the edges to the center of the output laser beam alongthe horizontal axis. In these instances, the intensity of the angularlydeflected laser beam at the center of the output beam may range from0.1% to about 99% of the intensity of the angularly deflected laserbeams at the edge of the output laser beam along the horizontal axis,such as from 0.5% to about 95%, such as from 1% to about 90%, such asfrom about 2% to about 85%, such as from about 3% to about 80%, such asfrom about 4% to about 75%, such as from about 5% to about 70%, such asfrom about 6% to about 65%, such as from about 7% to about 60%, such asfrom about 8% to about 55% and including from about 10% to about 50% ofthe intensity of the angularly deflected laser beams at the edge of theoutput laser beam along the horizontal axis. In other embodiments, thecontroller has a processor having memory operably coupled to theprocessor such that the memory includes instructions stored thereon,which when executed by the processor, cause the processor to produce anoutput laser beam having an increasing intensity from the edges to thecenter of the output laser beam along the horizontal axis. In theseinstances, the intensity of the angularly deflected laser beam at theedges of the output beam may range from 0.1% to about 99% of theintensity of the angularly deflected laser beams at the center of theoutput laser beam along the horizontal axis, such as from 0.5% to about95%, such as from 1% to about 90%, such as from about 2% to about 85%,such as from about 3% to about 80%, such as from about 4% to about 75%,such as from about 5% to about 70%, such as from about 6% to about 65%,such as from about 7% to about 60%, such as from about 8% to about 55%and including from about 10% to about 50% of the intensity of theangularly deflected laser beams at the center of the output laser beamalong the horizontal axis. In yet other embodiments, the controller hasa processor having memory operably coupled to the processor such thatthe memory includes instructions stored thereon, which when executed bythe processor, cause the processor to produce an output laser beamhaving an intensity profile with a Gaussian distribution along thehorizontal axis. In still other embodiments, the controller has aprocessor having memory operably coupled to the processor such that thememory includes instructions stored thereon, which when executed by theprocessor, cause the processor to produce an output laser beam having atop hat intensity profile along the horizontal axis.

In embodiments, light beam generators of interest may be configured toproduce angularly deflected laser beams in the output laser beam thatare spatially separated. Depending on the applied radiofrequency drivesignals and desired irradiation profile of the output laser beam, theangularly deflected laser beams may be separated by 0.001 μm or more,such as by 0.005 μm or more, such as by 0.01 μm or more, such as by 0.05μm or more, such as by 0.1 μm or more, such as by 0.5 μm or more, suchas by 1 μm or more, such as by 5 μm or more, such as by 10 μm or more,such as by 100 μm or more, such as by 500 μm or more, such as by 1000 μmor more and including by 5000 μm or more. In some embodiments, systemsare configured to produce angularly deflected laser beams in the outputlaser beam that overlap, such as with an adjacent angularly deflectedlaser beam along a horizontal axis of the output laser beam. The overlapbetween adjacent angularly deflected laser beams (such as overlap ofbeam spots) may be an overlap of 0.001 μm or more, such as an overlap of0.005 μm or more, such as an overlap of 0.01 μm or more, such as anoverlap of 0.05 μm or more, such as an overlap of 0.1 μm or more, suchas an overlap of 0.5 μm or more, such as an overlap of 1 μm or more,such as an overlap of 5 μm or more, such as an overlap of 10 μm or moreand including an overlap of 100 μm or more.

In certain instances, light beam generators configured to generate twoor more beams of frequency shifted light include laser excitationmodules as described in U.S. Pat. Nos. 9,423,353; 9,784,661 and10,006,852 and U.S. Patent Publication Nos. 2017/0133857 and2017/0350803, the disclosures of which are herein incorporated byreference.

In embodiments, particle sorters include a detector for detecting lightfrom the interrogation point of the flow cell that comprises a lightdetection system. Light detection systems of interest may include one ormore photodetectors. Photodetectors of interest may include, but are notlimited to optical sensors, such as active-pixel sensors (APSs),avalanche photodiode, image sensors, charge-coupled devices (CODs),intensified charge-coupled devices (ICCDs), light emitting diodes,photon counters, bolometers, pyroelectric detectors, photoresistors,photovoltaic cells, photodiodes, photomultiplier tubes,phototransistors, quantum dot photoconductors or photodiodes andcombinations thereof, among other photodetectors. In certainembodiments, light from the interrogation point is measured with acharge-coupled device (CCD), semiconductor charge-coupled devices (CCD),active pixel sensors (APS), complementary metal-oxide semiconductor(CMOS) image sensors or N-type metal-oxide semiconductor (NMOS) imagesensors.

In some embodiments, light detectors of interest include a plurality ofphotodetectors. In some instances, the light detector includes aplurality of solid-state detectors such as photodiodes. In certaininstances, the light detector includes a photodetector array, such as anarray of photodiodes. In these embodiments, the photodetector array mayinclude 4 or more photodetectors, such as 10 or more photodetectors,such as 25 or more photodetectors, such as 50 or more photodetectors,such as 100 or more photodetectors, such as 250 or more photodetectors,such as 500 or more photodetectors, such as 750 or more photodetectorsand including 1000 or more photodetectors. For example, the detector maybe a photodiode array having 4 or more photodiodes, such as 10 or morephotodiodes, such as 25 or more photodiodes, such as 50 or morephotodiodes, such as 100 or more photodiodes, such as 250 or morephotodiodes, such as 500 or more photodiodes, such as 750 or morephotodiodes and including 1000 or more photodiodes.

The photodetectors may be arranged in any geometric configuration asdesired, where arrangements of interest include, but are not limited toa square configuration, rectangular configuration, trapezoidalconfiguration, triangular configuration, hexagonal configuration,heptagonal configuration, octagonal configuration, nonagonalconfiguration, decagonal configuration, dodecagonal configuration,circular configuration, oval configuration as well as irregularpatterned configurations. The photodetectors in the photodetector arraymay be oriented with respect to the other (as referenced in an X-Zplane) at an angle ranging from 10° to 180°, such as from 15° to 170°,such as from 20° to 160°, such as from 25° to 150°, such as from 30° to120° and including from 45° to 90°. The photodetector array may be anysuitable shape and may be a rectilinear shape, e.g., squares,rectangles, trapezoids, triangles, hexagons, etc., curvilinear shapes,e.g., circles, ovals, as well as irregular shapes, e.g., a parabolicbottom portion coupled to a planar top portion. In certain embodiments,the photodetector array has a rectangular-shaped active surface.

Each photodetector (e.g., photodiode) in the array may have an activesurface with a width that ranges from 5 μm to 250 μm, such as from 10 μmto 225 μm, such as from 15 μm to 200 μm, such as from 20 μm to 175 μm,such as from 25 μm to 150 μm, such as from 30 μm to 125 μm and includingfrom 50 μm to 100 μm and a length that ranges from 5 μm to 250 μm, suchas from 10 μm to 225 μm, such as from 15 μm to 200 μm, such as from 20μm to 175 μm, such as from 25 μm to 150 μm, such as from 30 μm to 125 μmand including from 50 μm to 100 μm, where the surface area of eachphotodetector (e.g., photodiode) in the array ranges from 25 to μm² to10000 μm², such as from 50 to μm² to 9000 μm², such as from 75 to μm² to8000 μm², such as from 100 to μm² to 7000 μm², such as from 150 to μm²to 6000 μm² and including from 200 to μm² to 5000 μm².

The size of the photodetector array may vary depending on the amount andintensity of the light, the number of photodetectors and the desiredsensitivity and may have a length that ranges from 0.01 mm to 100 mm,such as from 0.05 mm to 90 mm, such as from 0.1 mm to 80 mm, such asfrom 0.5 mm to 70 mm, such as from 1 mm to 60 mm, such as from 2 mm to50 mm, such as from 3 mm to 40 mm, such as from 4 mm to 30 mm andincluding from 5 mm to 25 mm. The width of the photodetector array mayalso vary, ranging from 0.01 mm to 100 mm, such as from 0.05 mm to 90mm, such as from 0.1 mm to 80 mm, such as from 0.5 mm to 70 mm, such asfrom 1 mm to 60 mm, such as from 2 mm to 50 mm, such as from 3 mm to 40mm, such as from 4 mm to 30 mm and including from 5 mm to 25 mm. Assuch, the active surface of the photodetector array may range from 0.1mm² to 10000 mm², such as from 0.5 mm² to 5000 mm², such as from 1 mm²to 1000 mm², such as from 5 mm² to 500 mm², and including from 10 mm² to100 mm².

Photodetectors of interest are configured to measure collected light atone or more wavelengths, such as at 2 or more wavelengths, such as at 5or more different wavelengths, such as at 10 or more differentwavelengths, such as at 25 or more different wavelengths, such as at 50or more different wavelengths, such as at 100 or more differentwavelengths, such as at 200 or more different wavelengths, such as at300 or more different wavelengths and including measuring light emittedby a sample in the flow stream at 400 or more different wavelengths.

In some embodiments, photodetectors are configured to measure collectedlight over a range of wavelengths (e.g., 200 nm-1000 nm). In certainembodiments, photodetectors of interest are configured to collectspectra of light over a range of wavelengths. For example, systems mayinclude one or more detectors configured to collect spectra of lightover one or more of the wavelength ranges of 200 nm-1000 nm. In yetother embodiments, detectors of interest are configured to measure lightfrom the sample in the flow stream at one or more specific wavelengths.For example, systems may include one or more detectors configured tomeasure light at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm,605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm,710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm and any combinationsthereof. In certain embodiments, photodetectors may be configured to bepaired with specific fluorophores, such as those used with the sample ina fluorescence assay. In some embodiments, photodetectors are configuredto measure collected light across the entire fluorescence spectrum ofeach fluorophore in the sample.

The detector is configured to measure light continuously or in discreteintervals. In some instances, detectors of interest are configured totake measurements of the collected light continuously. In otherinstances, the light detector is configured to take measurements indiscrete intervals, such as measuring light every 0.001 millisecond,every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond,every 10 milliseconds, every 100 milliseconds and including every 1000milliseconds, or some other interval.

In some instances, the light detector also includes an opticaladjustment component. In some instances, optical adjustment is amagnification protocol configured to increase the size of the field oflight captured by the detector, such as by 5% or greater, such as by 10%or greater, such as by 25% or greater, such as by 50% or greater andincluding increasing the field of light captured by the detector by 75%or greater. In other instances, optical adjustment is a de-magnificationprotocol configured to decrease the field of light captured by thedetector, such as by 5% or greater, such as by 10% or greater, such asby 25% or greater, such as by 50% or greater and including decreasingthe field of light captured by the detector by 75% or greater. Incertain embodiments, optical adjustment is a focusing protocolconfigured to focus the light collected by the detector, such as byfocusing the beam of collected light by 5% or greater, such as by 10% orgreater, such as by 25% or greater, such as by 50% or greater andincluding focusing the beam of collected light by 75% or greater.

Optical adjustment components may be any convenient device or structurewhich provides the desired change in the collected light beam and mayinclude but is not limited to lenses, mirrors, pinholes, slits,gratings, light refractors, and any combinations thereof. The detectormay include one or more optical adjustment components as needed, such astwo or more, such as three or more, such as four or more and includingfive or more optical adjustment components. In certain embodiments, thedetector includes a focusing lens. The focusing lens, for example may bea de-magnifying lens. In other instances, the focusing lens is amagnifying lens. In other embodiments, the detector includes acollimator.

In certain embodiments, particle sorters include a combination ofdifferent optical adjustment components, such as a combination ofpinholes, lenses, mirrors, slits, etc. For example, in some embodiments,particle sorters include a focusing lens and a collimating lens. Inother embodiments, particle sorters include a collimating mirror and afocusing lens. In yet other embodiments, particle sorters include afocusing lens and a pinhole structure. In still other embodiments,particle sorters include a collimating lens and a pinhole structure. Instill other embodiments, particle sorters include a collimating lens anda slit structure.

In some embodiments, the detector and the optical adjustment componentare in optical communication but are not physically in contact.Depending on the size of the detector, the optical adjustment componentmay be positioned 0.05 mm or more from the detector, 0.1 mm or more,such as 0.5 mm or more, such as 1 mm or more, such as 10 mm or more,such as 25 mm or more, such as 50 mm or more, such as 100 mm or more,such as 250 mm or more, including 500 mm or more. In other embodiments,the optical adjustment component is physically coupled to the detector,such as with an adhesive, co-molded together or integrated together in ahousing having the optical adjustment component positioned adjacent tothe detector. As such, the optical adjustment component and detector maybe integrated into a single unit.

In some embodiments, the optical adjustment component is a focusing lenshaving a magnification ratio of from 0.1 to 0.95, such as amagnification ratio of from 0.2 to 0.9, such as a magnification ratio offrom 0.3 to 0.85, such as a magnification ratio of from 0.35 to 0.8,such as a magnification ratio of from 0.5 to 0.75 and including amagnification ratio of from 0.55 to 0.7, for example a magnificationratio of 0.6. For example, the focusing lens is, in certain instances, adouble achromatic de-magnifying lens having a magnification ratio ofabout 0.6. Depending on the distance between the detector and the lens,the surface area of the detector active surface, the focal length of thefocusing lens may vary, ranging from 5 mm to 20 mm, such as from 6 mm to19 mm, such as from 7 mm to 18 mm, such as from 8 mm to 17 mm, such asfrom 9 mm to 16 and including a focal length ranging from 10 mm to 15mm. In certain embodiments, the focusing lens has a focal length ofabout 13 mm.

In certain embodiments, optical adjustment components include one ormore fiber optics which are configured to relay light from theinterrogation point of the flow cell to the detector. Suitable fiberoptics for propagating light from the interrogation point of the flowcell to the active surface of the detector include, but are not limitedto, flow cytometer fiber optics systems such as those described in U.S.Pat. No. 6,809,804, the disclosure of which is herein incorporated byreference.

In other embodiments, detectors of interest are coupled to a collimator.The collimator may be any convenient collimating protocol, such as oneor more mirrors or curved lenses or a combination thereof. For example,the collimator is, in certain instances, a single collimating lens. Inother instances, the collimator is a collimating mirror. In yet otherinstances, the collimator includes a series of two or more lenses, suchas three or more lenses and including four or more lenses. In stillother instances, the collimator includes a mirror and a lens. Where thecollimator includes one or more lenses, the focal length of thecollimating lens may vary, ranging from 5 mm to 40 mm, such as from 6 mmto 37.5 mm, such as from 7 mm to 35 mm, such as from 8 mm to 32.5 mm,such as from 9 mm to 30 mm, such as from 10 mm to 27.5 mm, such as from12.5 mm to 25 mm and including a focal length ranging from 15 mm to 20mm.

In certain embodiments, the optical adjustment component is a wavelengthseparator. As discussed above, wavelength separators of interest referto an optical protocol for separating polychromatic light into itscomponent wavelengths for detection. Wavelength separation, according tocertain embodiments, may include selectively passing or blockingspecific wavelengths or wavelength ranges of the polychromatic light. Toseparate wavelengths of light, the light emitted by a sample in the flowstream may be passed through any convenient wavelength separatingprotocol, including but not limited to colored glass, bandpass filters,interference filters, dichroic mirrors, diffraction gratings,monochromators and combinations thereof, among other wavelengthseparating protocols. Particle sorters may include one or morewavelength separators, such as two or more, such as three or more, suchas four or more, such as five or more and including 10 or morewavelength separators. In one example, detectors include one bandpassfilter. In another example, detectors include two or more bandpassfilters. In another example, detectors include two or more bandpassfilters and a diffraction grating. In yet another example, detectorsinclude a monochromator. In certain embodiments, detectors include aplurality of bandpass filters and diffraction gratings configured into afilter wheel setup. Where detectors include two or more wavelengthseparators, the wavelength separators may be utilized individually or inseries to separate polychromatic light into component wavelengths. Insome embodiments, wavelength separators are arranged in series. In otherembodiments, wavelength separators are arranged individually such thatone or more measurements are conducted using each of the wavelengthseparators.

In some embodiments, detectors include one or more optical filters, suchas one or more bandpass filters. For example, optical filters ofinterest may include bandpass filters having minimum bandwidths rangingfrom 2 nm to 100 nm, such as from 3 nm to 95 nm, such as from 5 nm to 95nm, such as from 10 nm to 90 nm, such as from 12 nm to 85 nm, such asfrom 15 nm to 80 nm and including bandpass filters having minimumbandwidths ranging from 20 nm to 50 nm. In other embodiments, thewavelength separator is a diffraction grating. Diffraction gratings mayinclude, but are not limited to transmission, dispersive or reflectivediffraction gratings. Suitable spacings of the diffraction grating mayvary depending on the configuration of the flow cell nozzle chamber,detector and other optical adjust protocols present (e.g., focusinglens), ranging from 0.01 μm to 10 μm, such as from 0.025 μm to 7.5 μm,such as from 0.5 μm to 5 μm, such as from 0.75 μm to 4 μm, such as from1 μm to 3.5 μm and including from 1.5 μm to 3.5 μm.

FIG. 6 shows a flow cytometric particle sorter 600 in accordance with anillustrative embodiment of the present invention. The particle sorter600 includes a flow cytometer 610, a controller/processor 690 and amemory 695. The flow cytometer 610 includes one or more excitationlasers 615 a-615 c, a focusing lens 620, a flow chamber (i.e., a flowcell) 625, a forward scatter detector 630, a side scatter detector 635,a fluorescence collection lens 640, one or more beam splitters 645 a-645g, one or more bandpass filters 650 a-650 e, one or more longpass (“LP”)filters 655 a-655 b, and one or more fluorescent detectors 660 a-660 f.

The excitation lasers 615 a-c emit light in the form of a laser beam.The wavelengths of the laser beams emitted from excitation lasers 615a-615 c are 488 nm, 633 nm, and 325 nm, respectively, in the exampleparticle sorter of FIG. 6. The laser beams are first directed throughone or more of beam splitters 645 a and 645 b. Beam splitter 645 atransmits light at 488 nm and reflects light at 633 nm. Beam splitter645 b transmits UV light (light with a wavelength in the range of 10 to400 nm) and reflects light at 488 nm and 633 nm.

The laser beams are then directed to a focusing lens 620, which focusesthe beams onto the portion of a fluid stream where particles of a sampleare located, within the flow chamber 625. The flow chamber is part of afluidics system which directs particles, typically one at a time, in astream to the focused laser beam for interrogation. The flow chamber cancomprise a flow cell in a benchtop cytometer or a nozzle tip in astream-in-air cytometer.

The light from the laser beam(s) interacts with the particles in thesample by diffraction, refraction, reflection, scattering, andabsorption with re-emission at various different wavelengths dependingon the characteristics of the particle such as its size, internalstructure, and the presence of one or more fluorescent moleculesattached to or naturally present on or in the particle. The fluorescenceemissions as well as the diffracted light, refracted light, reflectedlight, and scattered light may be routed to one or more of the forwardscatter detector 630, the side scatter detector 635, and the one or morefluorescent detectors 660 a-660 f through one or more of the beamsplitters 645 a-645 g, the bandpass filters 650 a-650 e, the longpassfilters 655 a-655 b, and the fluorescence collection lens 640.

The fluorescence collection lens 640 collects light emitted from theparticle-laser beam interaction and routes that light towards one ormore beam splitters and filters. Bandpass filters, such as bandpassfilters 650 a-650 e, allow a narrow range of wavelengths to pass throughthe filter. For example, bandpass filter 650 a is a 510/20 filter. Thefirst number represents the center of a spectral band. The second numberprovides a range of the spectral band. Thus, a 510/20 filter extends 10nm on each side of the center of the spectral band, or from 500 nm to520 nm. Shortpass filters transmit wavelengths of light equal to orshorter than a specified wavelength. Longpass filters, such as longpassfilters 655 a-655 b, transmit wavelengths of light equal to or longerthan a specified wavelength of light. For example, longpass filter 655a, which is a 670 nm longpass filter, transmits light equal to or longerthan 670 nm. Filters are often selected to optimize the specificity of adetector for a particular fluorescent dye. The filters can be configuredso that the spectral band of light transmitted to the detector is closeto the emission peak of a fluorescent dye.

Beam splitters direct light of different wavelengths in differentdirections. Beam splitters can be characterized by filter propertiessuch as shortpass and longpass. For example, beam splitter 645 g is a620 SP beam splitter, meaning that the beam splitter 645 g transmitswavelengths of light that are 620 nm or shorter and reflects wavelengthsof light that are longer than 620 nm in a different direction. In oneembodiment, the beam splitters 645 a-645 g can comprise optical mirrors,such as dichroic mirrors.

The forward scatter detector 630 is positioned slightly off axis fromthe direct beam through the flow cell and is configured to detectdiffracted light, the excitation light that travels through or aroundthe particle in mostly a forward direction. The intensity of the lightdetected by the forward scatter detector is dependent on the overallsize of the particle. The forward scatter detector can include aphotodiode. The side scatter detector 635 is configured to detectrefracted and reflected light from the surfaces and internal structuresof the particle and tends to increase with increasing particlecomplexity of structure. The fluorescence emissions from fluorescentmolecules associated with the particle can be detected by the one ormore fluorescent detectors 660 a-660 f. The side scatter detector 635and fluorescent detectors can include photomultiplier tubes. The signalsdetected at the forward scatter detector 630, the side scatter detector635 and the fluorescent detectors can be converted to electronic signals(voltages) by the detectors. This data can provide information about thesample.

One of skill in the art will recognize that a flow cytometer inaccordance with an embodiment of the present invention is not limited tothe flow cytometer depicted in FIG. 6, but can include any flowcytometer known in the art. For example, a flow cytometer may have anynumber of lasers, beam splitters, filters, and detectors at variouswavelengths and in various different configurations.

In operation, cytometer operation is controlled by acontroller/processor 690, and the measurement data from the detectorscan be stored in the memory 695 and processed by thecontroller/processor 690. Although not shown explicitly, thecontroller/processor 690 is coupled to the detectors to receive theoutput signals therefrom and may also be coupled to electrical andelectromechanical components of the flow cytometer 600 to control thelasers, fluid flow parameters, and the like. Input/output (I/O)capabilities 697 may be provided also in the system. The memory 695,controller/processor 690, and I/O 697 may be entirely provided as anintegral part of the flow cytometer 610. In such an embodiment, adisplay may also form part of the I/O capabilities 697 for presentingexperimental data to users of the cytometer 600. Alternatively, some orall of the memory 695 and controller/processor 690 and I/O capabilitiesmay be part of one or more external devices such as a general purposecomputer. In some embodiments, some or all of the memory 695 andcontroller/processor 690 can be in wireless or wired communication withthe cytometer 610. The controller/processor 690 in conjunction with thememory 695 and the I/O 697 can be configured to perform variousfunctions related to the preparation and analysis of a flow cytometerexperiment.

The system illustrated in FIG. 6 includes six different detectors thatdetect fluorescent light in six different wavelength bands (which may bereferred to herein as a “filter window” for a given detector) as definedby the configuration of filters and/or splitters in the beam path fromthe flow cell 625 to each detector. Different fluorescent molecules usedfor a flow cytometer experiment will emit light in their owncharacteristic wavelength bands. The particular fluorescent labels usedfor an experiment and their associated fluorescent emission bands may beselected to generally coincide with the filter windows of the detectors.However, as more detectors are provided, and more labels are utilized,perfect correspondence between filter windows and fluorescent emissionspectra is not possible. It is generally true that although the peak ofthe emission spectra of a particular fluorescent molecule may lie withinthe filter window of one particular detector, some of the emissionspectra of that label will also overlap the filter windows of one ormore other detectors. This may be referred to as spillover. The I/O 697can be configured to receive data regarding a flow cytometer experimenthaving a panel of fluorescent labels and a plurality of cell populationshaving a plurality of markers, each cell population having a subset ofthe plurality of markers. The I/O 697 can also be configured to receivebiological data assigning one or more markers to one or more cellpopulations, marker density data, emission spectrum data, data assigninglabels to one or more markers, and cytometer configuration data. Flowcytometer experiment data, such as label spectral characteristics andflow cytometer configuration data can also be stored in the memory 695.The controller/processor 690 can be configured to evaluate one or moreassignments of labels to markers.

In certain embodiments, the subject particle sorters are flow cytometricsystems employing the above droplet deflector configured to apply aknown offset deflection force. In certain embodiments, the subjectparticle sorters are flow cytometric systems. Suitable flow cytometrysystems may include, but are not limited to, those described in Ormerod(ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997);Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in MolecularBiology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed.,Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. January; 49(pt1):17-28; Linden, et. al., Semin Throm Hemost. 2004 October;30(5):502-11; Alison, et al. J Pathol, 2010 December; 222(4):335-344;and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst.24(3):203-255; the disclosures of which are incorporated herein byreference. In certain instances, flow cytometry systems of interestinclude BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flowcytometer, BD Biosciences FACSCelesta™ flow cytometer, BD BiosciencesFACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BDBiosciences FACSymphony™ flow cytometer BD Biosciences LSRFortessa™ flowcytometer, BD Biosciences LSRFortess™ X-20 flow cytometer and BDBiosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cellsorter, BD Biosciences FACSLyric™ cell sorter and BD Biosciences Via™cell sorter BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™cell sorter, BD Biosciences Aria™ cell sorters and BD BiosciencesFACSMelody™ cell sorter, or the like. In some instances, the cell sorteris a BD FACSymphony™ S6 cell sorter; BD FACSMelody™ cell sorter; BDFACSAria™ III cell sorter; BD FACSAria™ Fusion cell sorter; BD FACSJazz™or BD Influx™ cell sorter.

In certain instances, the subject particle sorters are flow cytometrysystems configured for imaging particles in a flow stream byfluorescence imaging using radiofrequency tagged emission (FIRE), suchas those described in Diebold, et al. Nature Photonics Vol. 7(10);806-810 (2013) as well as described in U.S. Pat. Nos. 9,423,353;9,784,661; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316;10,288,546; 10,324,019; 10,408,758; 10,451,538; 10,620,111; and U.S.Patent Publication Nos. 2017/0133857; 2017/0328826; 2017/0350803;2018/0275042; 2019/0376895 and 2019/0376894 the disclosures of which areherein incorporated by reference.

In some embodiments, the subject systems are flow cytometric systems,such those described in U.S. Pat. Nos. 10,663,476; 10,620,111;10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074;10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341;9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034;8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875;7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692;5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766; thedisclosures of which are herein incorporated by reference in theirentirety.

Methods for Deflecting Droplets of a Droplet Stream by Applying A KnownOffset Deflection Force

Aspects of the present disclosure include methods for deflectingdroplets of a droplet stream with a droplet deflector configured toapply a known offset deflection force to the droplet stream. Asdescribed in greater detail above, by applying an “offset deflectionforce,” it is meant, for example, that the deflection force whichdiverts droplets in the droplet stream may comprise both a“side-to-side” direction and a “front-to-back” direction within ahorizontal plane that is orthogonal to the droplet stream, and in somecases, the deflection force is offset by applying the deflection forceafter rotating the angle at which the deflection force is applied to thedroplet stream around the longitudinal axis of the droplet stream. Asdescribed above, by applying a “known offset deflection force,” it ismeant applying a deflection force to droplets of a droplet stream thatis offset by design or a predetermined amount.

Methods according to certain embodiments include irradiating with alight source an interrogation point of a flow cell, detecting light fromthe interrogation point with a detector, producing a droplet stream fromfluid exiting the flow cell with a droplet generator, and deflectingdroplets of the droplet stream with a droplet deflector configured toapply a known offset deflection force to the droplet stream. Deflectingdroplets, including to sort particles, such as cells, according to thesubject methods results in increased sorting efficiency, such that fewerparticles of a sample are wasted (i.e., in advertently deflectingdroplets comprising particles, such as cells, into an unintended dropletdeposition position such that they go unsorted) when sorting a sample.In some cases, the efficiency of sorting may be improved such that morevariations of cell phenotypes may be collected and sorted when thesubject methods are employed. When used as part of flow cytometricallysorting a sample, the subject methods can improve the yield of particlesorting.

In certain embodiments, the droplet stream comprises a sample that is abiological sample and methods include sorting and collecting two or moredifferent types of cells, such that, in practicing the subject methods,a sample having particles is irradiated with a light source and lightfrom the sample is detected with a light detection system having one ormore photodetectors. In some embodiments, the sample is a biologicalsample. The term “biological sample” is used in its conventional senseto refer to a whole organism, plant, fungi or a subset of animaltissues, cells or component parts which may in certain instances befound in blood, mucus, lymphatic fluid, synovial fluid, cerebrospinalfluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cordblood, urine, vaginal fluid and semen. As such, a “biological sample”refers to both the native organism or a subset of its tissues as well asto a homogenate, lysate or extract prepared from the organism or asubset of its tissues, including but not limited to, for example,plasma, serum, spinal fluid, lymph fluid, sections of the skin,respiratory, gastrointestinal, cardiovascular, and genitourinary tracts,tears, saliva, milk, blood cells, tumors, organs. Biological samples maybe any type of organismic tissue, including both healthy and diseasedtissue (e.g., cancerous, malignant, necrotic, etc.). In certainembodiments, the biological sample is a liquid sample, such as blood orderivative thereof, e.g., plasma, tears, urine, semen, etc., where insome instances the sample is a blood sample, including whole blood, suchas blood obtained from venipuncture or fingerstick (where the blood mayor may not be combined with any reagents prior to assay, such aspreservatives, anticoagulants, etc.).

In certain embodiments the source of the sample is a “mammal” or“mammalian”, where these terms are used broadly to describe organismswhich are within the class Mammalia, including the orders carnivore(e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some instances,the subjects are humans. The methods may be applied to samples obtainedfrom human subjects of both genders and at any stage of development(i.e., neonates, infant, juvenile, adolescent, adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe present invention may be applied to samples from a human subject, itis to be understood that the methods may also be carried-out on samplesfrom other animal subjects (that is, in “non-human subjects”) such as,but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

In embodiments, an interrogation point of a flow cell is irradiated withlight from a light source. In practicing the subject methods, forexample, a sample (e.g., in a flow stream of a flow cytometer) may beirradiated in a flow cell with light from a light source. In someembodiments, the light source is a broadband light source, emittinglight having a broad range of wavelengths, such as for example, spanning50 nm or more, such as 100 nm or more, such as 150 nm or more, such as200 nm or more, such as 250 nm or more, such as 300 nm or more, such as350 nm or more, such as 400 nm or more and including spanning 500 nm ormore. For example, one suitable broadband light source emits lighthaving wavelengths from 200 nm to 1500 nm. Another example of a suitablebroadband light source includes a light source that emits light havingwavelengths from 400 nm to 1000 nm. Where methods include irradiatingwith a broadband light source, broadband light source protocols ofinterest may include, but are not limited to, a halogen lamp, deuteriumarc lamp, xenon arc lamp, stabilized fiber-coupled broadband lightsource, a broadband LED with continuous spectrum, superluminescentemitting diode, semiconductor light emitting diode, wide spectrum LEDwhite light source, a multi-LED integrated white light source, amongother broadband light sources or any combination thereof.

In other embodiments, methods includes irradiating with a narrow bandlight source emitting a particular wavelength or a narrow range ofwavelengths, such as for example with a light source which emits lightin a narrow range of wavelengths like a range of 50 nm or less, such as40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nmor less, such as 2 nm or less and including light sources which emit aspecific wavelength of light (i.e., monochromatic light). Where methodsinclude irradiating with a narrow band light source, narrow band lightsource protocols of interest may include, but are not limited to, anarrow wavelength LED, laser diode or a broadband light source coupledto one or more optical bandpass filters, diffraction gratings,monochromators or any combination thereof.

In certain embodiments, methods include irradiating the interrogationpoint with one or more lasers. As discussed above, the type and numberof lasers will vary depending on the sample as well as desired lightcollected and may be a gas laser, such as a helium-neon laser, argonlaser, krypton laser, xenon laser, nitrogen laser, CO₂ laser, CO laser,argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimerlaser, xenon chlorine (XeCl) excimer laser or xenon-fluorine (XeF)excimer laser or a combination thereof. In other instances, the methodsinclude irradiating the flow stream with a dye laser, such as astilbene, coumarin or rhodamine laser. In yet other instances, methodsinclude irradiating the flow stream with a metal-vapor laser, such as ahelium-cadmium (HeCd) laser, helium-mercury (HeHg) laser,helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontiumlaser, neon-copper (NeCu) laser, copper laser or gold laser andcombinations thereof. In still other instances, methods includeirradiating the flow stream with a solid-state laser, such as a rubylaser, an Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser,Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser, titanium sapphirelaser, thulim YAG laser, ytterbium YAG laser, ytterbium₂O₃ laser orcerium doped lasers and combinations thereof.

The sample may be irradiated with one or more of the above-mentionedlight sources, such as 2 or more light sources, such as 3 or more lightsources, such as 4 or more light sources, such as 5 or more lightsources and including 10 or more light sources. The light source mayinclude any combination of types of light sources. For example, in someembodiments, the methods include irradiating the interrogation point ofa flow cell with an array of lasers, such as an array having one or moregas lasers, one or more dye lasers and one or more solid-state lasers.

The sample may be irradiated with wavelengths ranging from 200 nm to1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm,such as from 350 nm to 900 nm and including from 400 nm to 800 nm. Forexample, where the light source is a broadband light source, theinterrogation point of the flow cell may be irradiated with wavelengthsfrom 200 nm to 900 nm. In other instances, where the light sourceincludes a plurality of narrow band light sources, the sample may beirradiated with specific wavelengths in the range from 200 nm to 900 nm.For example, the light source may be a plurality of narrow band LEDs (1nm-25 nm) each independently emitting light having a range ofwavelengths between 200 nm to 900 nm. In other embodiments, the narrowband light source includes one or more lasers (such as a laser array)and the sample is irradiated with specific wavelengths ranging from 200nm to 700 nm, such as with a laser array having gas lasers, excimerlasers, dye lasers, metal vapor lasers and solid-state laser asdescribed above.

Where more than one light source is employed, the interrogation point ofthe flow cell may be irradiated with the light sources simultaneously orsequentially, or a combination thereof. For example, the interrogationpoint of a flow cell may be simultaneously irradiated with each of thelight sources. In other embodiments, the interrogation point of the flowcell is sequentially irradiated with each of the light sources. Wheremore than one light source is employed to irradiate the interrogationpoint of the flow cell sequentially, the time each light sourceirradiates the interrogation point may independently be 0.001microseconds or more, such as 0.01 microseconds or more, such as 0.1microseconds or more, such as 1 microsecond or more, such as 5microseconds or more, such as 10 microseconds or more, such as 30microseconds or more and including 60 microseconds or more. For example,methods may include irradiating the sample with the light source (e.g.,laser) for a duration which ranges from 0.001 microseconds to 100microseconds, such as from 0.01 microseconds to 75 microseconds, such asfrom 0.1 microseconds to 50 microseconds, such as from 1 microsecond to25 microseconds and including from 5 microseconds to 10 microseconds. Inembodiments where the interrogation point is sequentially irradiatedwith two or more light sources, the duration the sample is irradiated byeach light source may be the same or different.

The time period between irradiation by each light source may also vary,as desired, being separated independently by a delay of 0.001microseconds or more, such as 0.01 microseconds or more, such as 0.1microseconds or more, such as 1 microsecond or more, such as 5microseconds or more, such as by 10 microseconds or more, such as by 15microseconds or more, such as by 30 microseconds or more and includingby 60 microseconds or more. For example, the time period betweenirradiation by each light source may range from 0.001 microseconds to 60microseconds, such as from 0.01 microseconds to 50 microseconds, such asfrom 0.1 microseconds to 35 microseconds, such as from 1 microsecond to25 microseconds and including from 5 microseconds to 10 microseconds. Incertain embodiments, the time period between irradiation by each lightsource is 10 microseconds. In embodiments where the interrogation pointof the flow cell is sequentially irradiated by more than two (i.e., 3 ormore) light sources, the delay between irradiation by each light sourcemay be the same or different.

The interrogation point of the flow cell may be irradiated continuouslyor in discrete intervals. In some instances, methods include irradiatingthe interrogation point of the flow cell with the light sourcecontinuously. In other instances, the interrogation point of the flowcell is irradiated with the light source in discrete intervals, such asirradiating every 0.001 millisecond, every 0.01 millisecond, every 0.1millisecond, every 1 millisecond, every 10 milliseconds, every 100milliseconds and including every 1000 milliseconds, or some otherinterval.

Depending on the light source, the interrogation point of the flow cellmay be irradiated from a distance which varies such as 0.01 mm or more,such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more,such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, suchas 10 mm or more, such as 15 mm or more, such as 25 mm or more andincluding 50 mm or more. Also, the angle of irradiation may also vary,ranging from 10° to 90°, such as from 15° to 85°, such as from 20° to80°, such as from 25° to 75° and including from 30° to 60°, for exampleat a 90° angle.

In certain embodiments, methods include irradiating the interrogationpoint of the flow cell with two or more beams of frequency shiftedlight. A light beam generator component may be employed having a laserand an acousto-optic device for frequency shifting the laser light. Inthese embodiments, methods include irradiating the acousto-optic devicewith the laser. Depending on the desired wavelengths of light producedin the output laser beam (e.g., for use in irradiating a sample in aflow stream), the laser may have a specific wavelength that varies from200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nmto 1000 nm, such as from 350 nm to 900 nm and including from 400 nm to800 nm. The acousto-optic device may be irradiated with one or morelasers, such as 2 or more lasers, such as 3 or more lasers, such as 4 ormore lasers, such as 5 or more lasers and including 10 or more lasers.The lasers may include any combination of types of lasers. For example,in some embodiments, the methods include irradiating the acousto-opticdevice with an array of lasers, such as an array having one or more gaslasers, one or more dye lasers and one or more solid-state lasers.

Where more than one laser is employed, the acousto-optic device may beirradiated with the lasers simultaneously or sequentially, or acombination thereof. For example, the acousto-optic device may besimultaneously irradiated with each of the lasers. In other embodiments,the acousto-optic device is sequentially irradiated with each of thelasers. Where more than one laser is employed to irradiate theacousto-optic device sequentially, the time each laser irradiates theacousto-optic device may independently be 0.001 microseconds or more,such as 0.01 microseconds or more, such as 0.1 microseconds or more,such as 1 microsecond or more, such as 5 microseconds or more, such as10 microseconds or more, such as 30 microseconds or more and including60 microseconds or more. For example, methods may include irradiatingthe acousto-optic device with the laser for a duration which ranges from0.001 microseconds to 100 microseconds, such as from 0.01 microsecondsto 75 microseconds, such as from 0.1 microseconds to 50 microseconds,such as from 1 microsecond to 25 microseconds and including from 5microseconds to 10 microseconds. In embodiments where the acousto-opticdevice is sequentially irradiated with two or more lasers, the durationthe acousto-optic device is irradiated by each laser may be the same ordifferent.

The time period between irradiation by each laser may also vary, asdesired, being separated independently by a delay of 0.001 microsecondsor more, such as 0.01 microseconds or more, such as 0.1 microseconds ormore, such as 1 microsecond or more, such as 5 microseconds or more,such as by 10 microseconds or more, such as by 15 microseconds or more,such as by 30 microseconds or more and including by 60 microseconds ormore. For example, the time period between irradiation by each lightsource may range from 0.001 microseconds to 60 microseconds, such asfrom 0.01 microseconds to 50 microseconds, such as from 0.1 microsecondsto 35 microseconds, such as from 1 microsecond to 25 microseconds andincluding from 5 microseconds to 10 microseconds. In certainembodiments, the time period between irradiation by each laser is 10microseconds. In embodiments where the acousto-optic device issequentially irradiated by more than two (i.e., 3 or more) lasers, thedelay between irradiation by each laser may be the same or different.

The acousto-optic device may be irradiated continuously or in discreteintervals. In some instances, methods include irradiating theacousto-optic device with the laser continuously. In other instances,the acousto-optic device is irradiated with the laser in discreteintervals, such as irradiating every 0.001 millisecond, every 0.01millisecond, every 0.1 millisecond, every 1 millisecond, every 10milliseconds, every 100 milliseconds and including every 1000milliseconds, or some other interval.

Depending on the laser, the acousto-optic device may be irradiated froma distance which varies such as 0.01 mm or more, such as 0.05 mm ormore, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm ormore, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm ormore, such as 15 mm or more, such as 25 mm or more and including 50 mmor more. Also, the angle of irradiation may also vary, ranging from 10°to 90°, such as from 15° to 85°, such as from 20° to 80°, such as from25° to 75° and including from 30° to 60°, for example at a 90° angle.

In embodiments, methods include applying radiofrequency drive signals tothe acousto-optic device to generate angularly deflected laser beams.Two or more radiofrequency drive signals may be applied to theacousto-optic device to generate an output laser beam with the desirednumber of angularly deflected laser beams, such as 3 or moreradiofrequency drive signals, such as 4 or more radiofrequency drivesignals, such as 5 or more radiofrequency drive signals, such as 6 ormore radiofrequency drive signals, such as 7 or more radiofrequencydrive signals, such as 8 or more radiofrequency drive signals, such as 9or more radiofrequency drive signals, such as 10 or more radiofrequencydrive signals, such as 15 or more radiofrequency drive signals, such as25 or more radiofrequency drive signals, such as 50 or moreradiofrequency drive signals and including 100 or more radiofrequencydrive signals.

The angularly deflected laser beams produced by the radiofrequency drivesignals each have an intensity based on the amplitude of the appliedradiofrequency drive signal. In some embodiments, methods includeapplying radiofrequency drive signals having amplitudes sufficient toproduce angularly deflected laser beams with a desired intensity. Insome instances, each applied radiofrequency drive signal independentlyhas an amplitude from about 0.001 V to about 500 V, such as from about0.005 V to about 400 V, such as from about 0.01 V to about 300 V, suchas from about 0.05 V to about 200 V, such as from about 0.1 V to about100 V, such as from about 0.5 V to about 75 V, such as from about 1 V to50 V, such as from about 2 V to 40 V, such as from 3 V to about 30 V andincluding from about 5 V to about 25 V. Each applied radiofrequencydrive signal has, in some embodiments, a frequency of from about 0.001MHz to about 500 MHz, such as from about 0.005 MHz to about 400 MHz,such as from about 0.01 MHz to about 300 MHz, such as from about 0.05MHz to about 200 MHz, such as from about 0.1 MHz to about 100 MHz, suchas from about 0.5 MHz to about 90 MHz, such as from about 1 MHz to about75 MHz, such as from about 2 MHz to about 70 MHz, such as from about 3MHz to about 65 MHz, such as from about 4 MHz to about 60 MHz andincluding from about 5 MHz to about 50 MHz.

In these embodiments, the angularly deflected laser beams in the outputlaser beam are spatially separated. Depending on the appliedradiofrequency drive signals and desired irradiation profile of theoutput laser beam, the angularly deflected laser beams may be separatedby 0.001 μm or more, such as by 0.005 μm or more, such as by 0.01 μm ormore, such as by 0.05 μm or more, such as by 0.1 μm or more, such as by0.5 μm or more, such as by 1 μm or more, such as by 5 μm or more, suchas by 10 μm or more, such as by 100 μm or more, such as by 500 μm ormore, such as by 1000 μm or more and including by 5000 μm or more. Insome embodiments, the angularly deflected laser beams overlap, such aswith an adjacent angularly deflected laser beam along a horizontal axisof the output laser beam. The overlap between adjacent angularlydeflected laser beams (such as overlap of beam spots) may be an overlapof 0.001 μm or more, such as an overlap of 0.005 μm or more, such as anoverlap of 0.01 μm or more, such as an overlap of 0.05 μm or more, suchas an overlap of 0.1 μm or more, such as an overlap of 0.5 μm or more,such as an overlap of 1 μm or more, such as an overlap of 5 μm or more,such as an overlap of 10 μm or more and including an overlap of 100 μmor more.

In certain instances, the flow stream is irradiated with a plurality ofangularly deflected beams of frequency-shifted light and a cell in theflow stream is imaged by fluorescence imaging using radiofrequencytagged emission (FIRE) to generate a frequency-encoded image, such asthose described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810(2013) as well as described in U.S. Pat. Nos. 9,423,353; 9,784,661;9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546;10,324,019; 10,408,758; 10,451,538; 10,620,111; and U.S. PatentPublication Nos. 2017/0133857; 2017/0328826; 2017/0350803; 2018/0275042;2019/0376895 and 2019/0376894 the disclosures of which are hereinincorporated by reference.

As discussed above, in embodiments light from the interrogation point ofthe flow cell is conveyed to a detector as described in greater detailbelow and may be measured by, in some embodiments, a plurality ofphotodetectors. In some embodiments, methods include measuring thecollected light over a range of wavelengths (e.g., 200 nm-1000 nm). Forexample, methods may include collecting spectra of light over one ormore of the wavelength ranges of 200 nm-1000 nm. In yet otherembodiments, methods include measuring collected light at one or morespecific wavelengths. For example, the collected light may be measuredat one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723nm, 780 nm, 785 nm, 647 nm, 617 nm and any combinations thereof. Incertain embodiments, methods including measuring wavelengths of lightwhich correspond to the fluorescence peak wavelength of fluorophores. Insome embodiments, methods include measuring collected light across theentire fluorescence spectrum of each fluorophore in a sample flowing ina flow stream.

The collected light may be measured continuously or in discreteintervals. In some instances, methods include taking measurements of thelight continuously. In other instances, the light is measured indiscrete intervals, such as measuring light every 0.001 millisecond,every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond,every 10 milliseconds, every 100 milliseconds and including every 1000milliseconds, or some other interval.

Measurements of the collected light may be taken one or more timesduring the subject methods, such as 2 or more times, such as 3 or moretimes, such as 5 or more times and including 10 or more times. Incertain embodiments, the light propagation is measured 2 or more times,with the data in certain instances being averaged.

Light from the interrogation point may be measured at one or morewavelengths of, such as at 5 or more different wavelengths, such as at10 or more different wavelengths, such as at 25 or more differentwavelengths, such as at 50 or more different wavelengths, such as at 100or more different wavelengths, such as at 200 or more differentwavelengths, such as at 300 or more different wavelengths and includingmeasuring the collected light at 400 or more different wavelengths.

Methods according to the present disclosure further comprise producing adroplet stream from fluid exiting the flow cell with a dropletgenerator. As described above, the droplet generator may be anyconvenient device for producing a droplet stream from fluid exiting theflow cell. In some instances, the droplet generator is an oscillatingtransducer, such as, for example, a piezo-oscillator.

Methods according to the present disclosure further comprise deflectingdroplets of the droplet stream with a droplet deflector configured toapply a known offset deflection force to the droplet stream. Asdescribed above, to sort target droplets, such as, for example, targetdroplets containing particles of interest, the analyzed droplet streamis subjected to a known offset deflection force by a droplet deflector(as described above).

As described in greater detail above, in some embodiments, the dropletdeflector comprises first and second plates configured to be offset fromone another. In such embodiments, the first and second plates may beconfigured to be adjustably offset from one another. In instances, thefirst and second plates may be configured to be adjustably offset fromone another with respect to a horizontal plane. In such instances, thehorizontal plane is perpendicular to the axis (i.e., the longitudinalaxis) of the droplet stream.

In embodiments, the first plate comprises an elongated sectionconfigured to allow the first plate to be adjustably offset from thesecond plate with respect to the horizontal plane. In such embodiments,the elongated section of the first plate comprises a set screwconfigured to allow the first plate to be adjustably offset from thesecond plate with respect to the horizontal plane.

As described above, in examples, the first and second plates areconfigured to be adjustably offset from each other by greater than 0 mmto 5 mm. In such examples, the first and second plates are configured tobe adjustably offset from each other in increments that are determinedbased on the threat pitch of a set screw configured to adjustably offsetthe first plate from the second plate.

In instances, the known offset deflection force is sufficient to offseta drop deposition position by 2 mm or more when measured at a distanceof 140 mm below the lowest point of the first deflection plate. As such,the known offset deflection force comprises components in both the“side-to-side” axis and “front-to-back” axis of a horizontal planeorthogonal to the longitudinal axis of the flow stream, as describedabove. In other instances, the known offset deflection force issufficient to offset a drop deposition position by one droplet diameteror less.

In some cases, the droplet deflector according to the present disclosureis configured to deflect droplets into a plurality of partitions. Insuch cases, the partitions may comprise a collection container. In othercases, the collection container may be a multi-well plate. In stillother cases, the multi-well plate contains 1536 or fewer wells. In someinstances, the partitions comprise collection tubes. In such instances,the diameter of each collection tube is 1.8 mm or less.

As described above, the droplet deflectors of the subject methods maycomprise first and second plates configured to be parallel to oneanother. That is, even when offset from each other, the first and secondplaces face one other in a parallel manner. In other cases, the firstand second plates are configured to be adjustably rotated to face oneanother. That is, as the offset between the first and second plates isadjusted, the first and second plates may be correspondingly rotatedsuch that the plates face each other.

In some cases, the second plate comprises an elongated sectionconfigured to allow the second plate to be adjustably offset from thefirst plate with respect to the horizontal plane. In such cases, theelongated section of the second plate may comprise a set screwconfigured to allow the second plate to be adjustably offset from thefirst plate with respect to the horizontal plane.

As described in greater detail above, in embodiments, the dropletdeflector further comprises an actuator, e.g., a motor, configured toadjust the offset between the first and second plates. In someembodiments, the actuator, e.g., motor, is operably linked to a feedbacksubsystem, as described above. In such embodiments, the feedbacksubsystem may comprise a controller operably connected to the actuator,e.g., motor, and to a detector configured to detect a distance a dropletof the droplet stream is offset. In still other embodiments, thefeedback subsystem is configured to iteratively adjust the offsetbetween the first and second plates.

In embodiments, the first and second plates are metallic. A voltageapplied to each of the metallic plates such that droplets flowingtherethrough are accelerated and deflected at a plurality of differentangles based on the charge and polarity of the charge of the droplet.The voltage applied to the first and second metallic plates to divertcharged particles may be 10 mV or more, such as 25 mV or more, such as50 mV or more, such as 100 mV or more, such as 250 mV or more, such as500 mV or more, such as 750 mV or more, such as 1000 mV or more, such as2500 mV or more, such as 5000 mV or more, such as 10000 V or more, suchas 15000 V or more, such as 25000 V or more, such as 50000 V or more andincluding 100000 V or more. In certain embodiments, the voltage appliedto the first and second metallic plates is from 0.5 kV to 15 kV, such asfrom 1 kV to 15 kV, such as from 1.5 kV to 12.5 kV and including from 2kV to 10 kV. As such, the electric field strength between the metallicplates ranges from 0.1 V/m to 1×10⁷ V/m, such as from 0.5 V/m to 5×10⁶,such as from 1 V/m to 1×10⁶ V/m, such as from 5 V/m to 5×10⁵ V/m, suchas from 10 V/m to 1×10⁵ V/m and including from 50 V/m to 5×10⁴ V/m, forexample 1×10⁵ V/m to 2×10⁶ V/m.

In instances, the metallic plates are spaced apart by 1 mm or more. Inother instances, the metallic plates are spaced apart by 3 mm or more.In examples, the first and second plates are rectangular.

In some embodiments, methods for sorting components of a sample includesorting particles (e.g., cells in a biological sample) with a particlesorting module such as described in U.S. Patent Publication No.2017/0299493, filed on Mar. 28, 2017, the disclosure of which isincorporated herein by reference. In certain embodiments, cells of thesample are sorted using a sort decision module having a plurality ofsort decision units, such as those described in U.S. Provisional PatentApplication No. 62/803,264, filed on Feb. 8, 2019, the disclosure ofwhich is incorporated herein by reference.

Particle Sorting Modules

Aspects of the present disclosure include a particle sorting module. Inembodiments, particle sorting modules include a droplet deflectordescribed herein that is configured to apply a known offset deflectionforce to a droplet stream and to, for example, divert dropletscontaining analyzed particles to a receiving location. As described ingreater detail above, by applying an “offset deflection force,” it ismeant, for example, that the deflection force which diverts droplets inthe droplet stream may comprise both a “side-to-side” direction and a“front-to-back” direction within a horizontal plane that is orthogonalto the droplet stream, and in some cases, the deflection force is offsetby applying the deflection force after rotating the angle at which thedeflection force is applied to the droplet stream around thelongitudinal axis of the droplet stream. As described above, by applyinga “known offset deflection force,” it is meant applying a deflectionforce to droplets of a droplet stream that is offset by design or apredetermined amount.

Diversion of a droplet of interest to a receiving location may beachieved by the droplet deflector via, for example, electrostaticcharging of the droplet and deflection of the charged droplet from theflow stream by the application of an electrostatic field. In suchinstances, the voltage applied to first and second plate of the dropletdeflector in the subject particle sorting modules may be 10 mV or more,such as 25 mV or more, such as 50 mV or more, such as 75 mV or more,such as 100 mV or more, such as 250 mV or more, such as 500 mV or more,such as 750 mV or more, such as 1 V or more, such as 2.5 V or more, suchas 5 V or more, such as 10 V or more, such as 25 V or more, such as 50 Vor more and including 100 V or more, such as 500 V or more, such as 1000V or more, such as 5000 V or more, such as 10000 V or more, such as15000 V or more, such as 25000 V or more, such as 50000 V or more andincluding 100000 V or more. In certain embodiments, the voltage appliedto each set of parallel metallic plates is from 0.5 kV to 15 kV, such asfrom 1 kV to 15 kV, such as from 1.5 kV to 12.5 kV and including from 2kV to 10 kV.

In embodiments, particle sorting modules according to the presentdisclosure may be for sorting components of a sample, such as cells in abiological sample. The term “sorting” is used herein in its conventionalsense to refer to separating components (e.g., cells, non-cellularparticles such as biological macromolecules) of the sample and in someinstances, as described below, delivering the separated components to areceiving location having one or more containers. For example, thesubject particle sorting modules may be configured for sorting sampleshaving 2 or more components, such as 3 or more components, such as 4 ormore components, such as 5 or more components, such as 10 or morecomponent, such as 15 or more components and including soring a samplehaving 25 or more components. One or more of the sample components maybe separated from the sample and delivered to a container, such as 2 ormore sample components, such as 3 or more sample components, such as 4or more sample components, such as 5 or more sample components, such as10 or more sample components and including 15 or more sample componentsmay be separated from the sample and delivered to a container at thereceiving location.

The particle sorting module may be configured to produce an analyzedstream of droplets and deflect each analyzed droplet from the stream ofanalyzed droplets to a deflected droplet receiving location (i.e.,droplet deposition location). As used herein, the term “deflecteddroplet receiving location” (i.e., droplet deposition location orposition) refers to a location downstream (with respect to the dropletstream) from the droplet deflector where a sorted droplet containing acell of interest may be collected after it has been deflected by thedroplet deflector. As described above, droplets in the flow stream maybe diverted from their normal trajectory along the longitudinal axis ofthe droplet stream by a distance by 0.001 mm or more as measuredradially across a plane orthogonal to the longitudinal axis of the flowstream, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mmor more, such as 2 mm or more, such as 5 mm or more, such as 10 mm ormore, such as 15 mm or more, such as 20 mm or more, such as 25 mm ormore, such as 30 mm or more, such as 35 mm or more and including 50 mmor more. For example, the droplets in the droplet stream may be divertedby a distance of from 0.001 mm to 100 mm, such as from 0.005 mm to 95mm, such as from 0.001 mm to 90 mm, such as from 0.05 mm to 85 mm, suchas from 0.01 mm to 80 mm, such as from 0.05 mm to 75 mm, such as from0.1 mm to 70 mm, such as from 0.5 mm to 65 mm, such as from 1 mm 60 mm,such as from 5 mm to 55 mm and including from 10 mm to 50 mm. As such,the droplet receiving location may be 0.001 mm or more from thelongitudinal axis of the flow stream, such as 0.005 mm or more, such as0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, suchas 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 5mm or more, such as 10 mm or more, such as 15 mm or more, such as 20 mmor more, such as 25 mm or more, such as 30 mm or more, such as 35 mm ormore and including 50 mm or more from the longitudinal axis of the flowstream. The droplet receiving location of the subject particle sortingmodules may be at a plurality of different offsets with respect to a“front-to-back” axis of a horizontal plane perpendicular to thelongitudinal axis of the flow stream, such as from one droplet diameteror less to 5 mm in the front to back axis, such as from one dropletdiameter or less to 4 mm, such as from two droplet diameters to 3 mm,including two and a half droplet diameters to 2.5 mm.

In embodiments, subject particle sorting modules include a dropletdeflector that comprises first and second plates configured to be offsetfrom one another. In some embodiments, the first and second plates areconfigured to be adjustably offset from one another. In instances, thefirst and second plates are configured to be adjustably offset from oneanother with respect to a horizontal plane. In other instances, thehorizontal plane is perpendicular to the axis of the droplet stream.

In some embodiments of the particle sorter module, the first platecomprises an elongated section configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane. In such embodiments, the elongated section of the first plate maycomprise a set screw configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane.

In examples, the first and second plates of the particle sorting moduleare configured to be adjustably offset from each other by greater than 0mm to 5 mm. In such examples, the first and second plates are configuredto be adjustably offset from each other in increments that aredetermined based on the threat pitch of a set screw configured toadjustably offset the first plate from the second plate.

In instances of the particle sorting module, the known offset deflectionforce is sufficient to offset a drop deposition position by 2 mm ormore, such as 2 mm or more when the offset is measured at a distance of140 mm below the lowest point of the first deflection plate. As such,the known offset deflection force comprises components in both the“side-to-side” axis and “front-to-back” axis of a horizontal planeorthogonal to the longitudinal axis of the flow stream, as describedabove. In other instances, the known offset deflection force issufficient to offset a drop deposition position by one droplet diameteror less.

In some cases, the droplet deflector of the particle sorting moduleaccording to the present disclosure is configured to deflect dropletsinto a plurality of partitions. In such cases, the partitions maycomprise a collection container. In other cases, the collectioncontainer may be a multi-well plate. In still other cases, themulti-well plate contains 1536 or fewer wells. In some instances, thepartitions comprise collection tubes. In such instances, the diameter ofeach collection tube is 1.8 mm or less.

As described above, the droplet deflectors of the subject particlesorting modules may comprise first and second plates configured to beparallel to one another. That is, even when offset from each other, thefirst and second plates remain oriented in a parallel configuration. Inother cases, the first and second plates are configured to be adjustablyrotated to face one another. That is, as the offset between the firstand second plates is adjusted, the first and second plates may becorrespondingly rotated such that the plates face each other.

In some cases, the second plate of the droplet deflector comprises anelongated section configured to allow the second plate to be adjustablyoffset from the first plate with respect to the horizontal plane. Insuch cases, the elongated section of the second plate may comprise a setscrew configured to allow the second plate to be adjustably offset fromthe first plate with respect to the horizontal plane.

As described in greater detail above, in embodiments, the dropletdeflector of the particle sorting module further comprises an actuator,e.g., a motor, configured to adjust the offset between the first andsecond plates. In some embodiments, the actuator, e.g., motor, isoperably linked to a feedback subsystem, as described above. In suchembodiments, the feedback subsystem may comprise a controller operablyconnected to the actuator, e.g., motor, and to a detector configured todetect a distance a droplet of the droplet stream is offset. In stillother embodiments, the feedback subsystem is configured to iterativelyadjust the offset between the first and second plates.

In embodiments, the particle sorting module comprises a dropletdeflector wherein the first and second plates are metallic. A voltagemay be applied to each of the metallic plates of the droplet deflectorsuch that droplets flowing therethrough are accelerated and deflected ata plurality of different angles based on the charge and polarity of thecharge of the droplet. The voltage applied to the first and secondmetallic plates to divert charged droplets may be 10 mV or more, such as25 mV or more, such as 50 mV or more, such as 100 mV or more, such as250 mV or more, such as 500 mV or more, such as 750 mV or more, such as1000 mV or more, such as 2500 mV or more, such as 5000 mV or more, suchas 10000 V or more, such as 15000 V or more, such as 25000 V or more,such as 50000 V or more and including 100000 V or more. In certainembodiments, the voltage applied to the first and second metallic platesis from 0.5 kV to 15 kV, such as from 1 kV to 15 kV, such as from 1.5 kVto 12.5 kV and including from 2 kV to 10 kV. As such, the electric fieldstrength between the metallic plates ranges from 0.1 V/m to 1×10⁷ V/m,such as from 0.5 V/m to 5×10⁶, such as from 1 V/m to 1×10⁶ V/m, such asfrom 5 V/m to 5×10⁵ V/m, such as from 10 V/m to 1×10⁵ V/m and includingfrom 50 V/m to 5×10⁴ V/m, for example 1×10⁵ V/m to 2×10⁶ V/m.

In instances, the metallic plates of the droplet deflector are spacedapart by 1 mm or more. In other instances, the metallic plates arespaced apart by 3 mm or more. In examples, the first and second platesare rectangular.

Utility

The subject particle sorter, methods and particle sorting module finduse in a variety of applications where it is desirable to sort particlecomponents, such as cells, in a sample in a fluid medium, such as abiological sample. In some embodiments, the particle sorters, methodsand particle sorter modules described herein find use in flow cytometrycharacterization of biological samples labeled with fluorescent tags. Inother embodiments, the particle sorters, methods and particle sortermodules find use in spectroscopy of emitted light. In addition, thesubject particle sorters, methods and particle sorter modules find usein improving the efficiency of sorting a sample (e.g., in a flowstream). By improving the efficiency of sorting a sample, it is meantthat fewer particles, such as cells, of a sample may be wasted (i.e.,disposing of particles such as cells such that they go unused) whensorting a sample when the subject particle sorters, methods and particlesorter modules are employed. In particular, the subject particle sortersand methods may improve efficiency of sorting and in particular reducethe number of cells wasted when droplets containing target particles,such as target cells, are deflected but are not received in thedesignated partition, such as a collection container, due a lack ofalignment between the deflected droplet and the partition resulting fromapplying a deflection force that is not a known offset deflection force.In certain instances, the efficiency of sorting may be improved suchthat more variations of particles may be collected and sorted when thesubject particle sorters, methods and particle sorter modules areemployed. By variations of particles, it is meant, for example, cellphenotypes, such that a larger number of different cell phenotypes aresorted when embodiments of the present disclosure are employed.Embodiments of the present disclosure find use where it is desirable toprovide a flow cytometer with improved cell sorting efficiency, enhancedparticle collection, particle charging efficiency, more accurateparticle charging and enhanced particle deflection during cell sorting.

Embodiments of the present disclosure also find use in applicationswhere cells prepared from a biological sample may be desired forresearch, laboratory testing or for use in therapy. In some embodiments,the subject methods and devices may facilitate obtaining individualcells prepared from a target fluidic or tissue biological sample. Forexample, the subject methods and systems facilitate obtaining cells fromfluidic or tissue samples to be used as a research or diagnosticspecimen for diseases such as cancer. Likewise, the subject methods andsystems may facilitate obtaining cells from fluidic or tissue samples tobe used in therapy. Methods and devices of the present disclosure allowfor separating and collecting cells from a biological sample (e.g.,organ, tissue, tissue fragment, fluid) with enhanced efficiency and lowcost as compared to traditional flow cytometry systems.

Notwithstanding the appended claims, the disclosure is also defined bythe following clauses:

1. A particle sorter comprising:

a flow cell;

a light source for irradiating an interrogation point of the flow cell;

a detector for detecting light from the interrogation point;

a droplet generator for producing a droplet stream from fluid exitingthe flow cell; and

a droplet deflector configured to apply a known offset deflection forceto the droplet stream.

2. The particle sorter according to clause 1, wherein the dropletdeflector comprises first and second plates configured to be offset fromone another.3. The particle sorter according to clause 2, wherein the first andsecond plates are configured to be adjustably offset from one another.4. The particle sorter according to clause 3, wherein the first andsecond plates are configured to be adjustably offset from one anotherwith respect to a horizontal plane.5. The particle sorter according to clause 4, wherein the horizontalplane is perpendicular to the axis of the droplet stream.6. The particle sorter according to clause 4, wherein the first platecomprises an elongated section configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane.7. The particle sorter according to clause 6, wherein the elongatedsection of the first plate comprises a set screw configured to allow thefirst plate to be adjustably offset from the second plate with respectto the horizontal plane.8. The particle sorter according to any of clauses 3-7, wherein thefirst and second plates are configured to be adjustably offset from eachother by greater than 0 mm to 5 mm.9. The particle sorter according to clause 7, wherein the first andsecond plates are configured to be adjustably offset from each other inincrements determined by the thread pitch of the set screw.10. The particle sorter according to any of clauses 1-9, wherein theknown offset deflection force is sufficient to offset a drop depositionposition by 2 mm or more.11. The particle sorter according to any of clauses 1-10, wherein theknown offset deflection force is sufficient to offset a drop depositionposition by one droplet diameter or less.12. The particle sorter according to any of clauses 1-11, furthercomprising a plurality of partitions configured to receive dropletsdeflected by the droplet deflector.13. The particle sorter according to clause 12, wherein the partitionscomprise a collection container.14. The particle sorter according to clause 13, wherein the collectioncontainer is a multi-well plate.15. The particle sorter according to clause 14, wherein the multi-wellplate contains 1536 or fewer wells.16. The particle sorter according to clause 12, wherein the partitionscomprise collection tubes.17. The particle sorter according to clause 12, wherein the diameter ofeach partition is 1.8 mm or less.18. The particle sorter according to any of clauses 2-9, wherein thefirst and second plates are configured to be parallel to one another.19. The particle sorter according to any of clauses 3-9, wherein thefirst and second plates are configured to be adjustably rotated to faceone another.20. The particle sorter according to clause 6, wherein the second platecomprises an elongated section configured to allow the second plate tobe adjustably offset from the first plate with respect to the horizontalplane.21. The particle sorter according to clause 20, wherein the elongatedsection of the second plate comprises a set screw configured to allowthe second plate to be adjustably offset from the first plate withrespect to the horizontal plane.22. The particle sorter according to any of clauses 3-9, wherein thedroplet deflector further comprises an actuator configured to adjust theoffset between the first and second plates.23. The particle sorter according to clause 22, wherein the actuator isoperably linked to a feedback subsystem.24. The particle sorter according to clause 23, wherein the feedbacksubsystem comprises a controller operably connected to the actuator andto a detector configured to detect a distance a droplet of the dropletstream is offset.25. The particle sorter according to any of clauses 23-24, wherein thefeedback subsystem is configured to iteratively adjust the offsetbetween the first and second plates.26. The particle sorter according to any of clauses 2-9, wherein thefirst and second plates are metallic.27. The particle sorter according to clause 26, wherein the metallicplates are spaced apart by 1 mm or more.28. The particle sorter according to clause 26, wherein the metallicplates are spaced apart by 3 mm or more.29. The particle sorter according to any of clauses 2-9, wherein thefirst and second plates are rectangular.30. The particle sorter according to any of clauses 1-29, wherein thelight source is a laser.31. A method comprising:

irradiating with a light source an interrogation point of a flow cell;

detecting light from the interrogation point with a detector;

producing a droplet stream from fluid exiting the flow cell with adroplet generator; and

deflecting droplets of the droplet stream with a droplet deflectorconfigured to apply a known offset deflection force to the dropletstream.

32. The method according to clause 31, wherein the droplet deflectorcomprises first and second plates configured to be offset from oneanother.33. The method according to clause 32, wherein the first and secondplates are configured to be adjustably offset from one another.34. The method according to clause 33, wherein the first and secondplates are configured to be adjustably offset from one another withrespect to a horizontal plane.35. The method according to clause 34, wherein the horizontal plane isperpendicular to the axis of the droplet stream.36. The method according to clause 34, wherein the first plate comprisesan elongated section configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane.37. The method according to clause 36, wherein the elongated section ofthe first plate comprises a set screw configured to allow the firstplate to be adjustably offset from the second plate with respect to thehorizontal plane.38. The method according to any of clauses 33-37, wherein the first andsecond plates are configured to be adjustably offset from each other bygreater than 0 mm to 5 mm.39. The method according to clause 37, wherein the first and secondplates are configured to be adjustably offset from each other inincrements determined by the thread pitch of the set screw.40. The method according to any of clauses 31-39, wherein the knownoffset deflection force is sufficient to offset a drop depositionposition by 2 mm or more.41. The method according to any of clauses 31-40, wherein the knownoffset deflection force is sufficient to offset a drop depositionposition by one droplet diameter or less.42. The method according to any of clauses 31-41, wherein the dropletdeflector is further configured to deflect droplets into a plurality ofpartitions.43. The method according to clause 42, wherein the partitions comprise acollection container.44. The method according to clause 43, wherein the collection containeris a multi-well plate.45. The method according to clause 44, wherein the multi-well platecontains 1536 or fewer wells.46. The method according to clause 42, wherein the partitions comprisecollection tubes.47. The method according to clause 42, wherein the diameter of eachpartition is 1.8 mm or less.48. The method according to any of clauses 32-39, wherein the first andsecond plates are configured to be parallel to one another.49. The method according to any of clauses 33-39, wherein the first andsecond plates are configured to be adjustably rotated to face oneanother.50. The method according to clause 36, wherein the second platecomprises an elongated section configured to allow the second plate tobe adjustably offset from the first plate with respect to the horizontalplane.51. The method according to clause 50, wherein the elongated section ofthe second plate comprises a set screw configured to allow the secondplate to be adjustably offset from the first plate with respect to thehorizontal plane.52. The method according to any of clauses 33-39, wherein the dropletdeflector further comprises an actuator configured to adjust the offsetbetween the first and second plates.53. The method according to clause 52, wherein the actuator is operablylinked to a feedback subsystem.54. The method according to clause 53, wherein the feedback subsystemcomprises a controller operably connected to the actuator and to adetector configured to detect a distance a droplet of the droplet streamis offset.55. The method according to any of clauses 53-54, wherein the feedbacksubsystem is configured to iteratively adjust the offset between thefirst and second plates.56. The method according to any of clauses 32-39, wherein the first andsecond plates are metallic.57. The method according to clause 56, wherein the metallic plates arespaced apart by 1 mm or more.58. The method according to clause 56, wherein the metallic plates arespaced apart by 3 mm or more.59. The method according to any of clauses 32-39, wherein the first andsecond plates are rectangular.60. The method according to any of clauses 31-59, wherein the flowstream is irradiated by a laser.61. A particle sorting module comprising a droplet deflector configuredto apply a known offset deflection force to a droplet stream.62. The particle sorting module according to clause 61, wherein thedroplet deflector comprises first and second plates configured to beoffset from one another.63. The particle sorting module according to clause 62, wherein thefirst and second plates are configured to be adjustably offset from oneanother.64. The particle sorting module according to clause 63, wherein thefirst and second plates are configured to be adjustably offset from oneanother with respect to a horizontal plane.

65. The particle sorting module according to clause 64, wherein thehorizontal plane is perpendicular to the axis of the droplet stream.

66. The particle sorting module according to clause 64, wherein thefirst plate comprises an elongated section configured to allow the firstplate to be adjustably offset from the second plate with respect to thehorizontal plane.67. The particle sorting module according to clause 66, wherein theelongated section of the first plate comprises a set screw configured toallow the first plate to be adjustably offset from the second plate withrespect to the horizontal plane.68. The particle sorting module according to any of clauses 63-67,wherein the first and second plates are configured to be adjustablyoffset from each other by greater than 0 mm to 5 mm.69. The particle sorting module according to clause 67, wherein thefirst and second plates are configured to be adjustably offset from eachother in increments determined by the thread pitch of the set screw.70. The particle sorting module according to any of clauses 61-69,wherein the known offset deflection force is sufficient to offset a dropdeposition position by 2 mm or more.71. The particle sorting module according to any of clauses 61-70,wherein the known offset deflection force is sufficient to offset a dropdeposition position by one droplet diameter or less.72. The particle sorting module according to any of clauses 61-71,wherein the droplet deflector is further configured to deflect dropletsinto a plurality of partitions.73. The particle sorting module according to clause 72, wherein thepartitions comprise a collection container.74. The particle sorting module according to clause 73, wherein thecollection container is a multi-well plate.75. The particle sorting module according to clause 74, wherein themulti-well plate contains 1536 or fewer wells.76. The particle sorting module according to clause 75, wherein thepartitions comprise collection tubes.77. The particle sorting module according to clause 72, wherein thediameter of each partition is 1.8 mm or less.78. The particle sorting module according to any of clauses 62-69,wherein the first and second plates are configured to be parallel to oneanother.79. The particle sorting module according to any of clauses 63-69,wherein the first and second plates are configured to be adjustablyrotated to face one another.80. The particle sorting module according to clause 66, wherein thesecond plate comprises an elongated section configured to allow thesecond plate to be adjustably offset from the first plate with respectto the horizontal plane.81. The particle sorting module according to clause 80, wherein theelongated section of the second plate comprises a set screw configuredto allow the second plate to be adjustably offset from the first platewith respect to the horizontal plane.82. The particle sorting module according to any of clauses 63-69,wherein the droplet deflector further comprises an actuator configuredto adjust the offset between the first and second plates.83. The particle sorting module according to clause 82, wherein theactuator is operably linked to a feedback subsystem.84. The particle sorting module according to clause 83, wherein thefeedback subsystem comprises a controller operably connected to theactuator and to a detector configured to detect a distance a droplet ofthe droplet stream is offset.85. The particle sorting module according to any of clauses 83-84,wherein the feedback subsystem is configured to iteratively adjust theoffset between the first and second plates.86. The particle sorting module according to any of clauses 62-69,wherein the first and second plates are metallic.87. The particle sorting module according to clause 86, wherein themetallic plates are spaced apart by 1 mm or more.88. The particle sorting module according to clause 86, wherein themetallic plates are spaced apart by 3 mm or more.89. The particle sorting module according to any of clauses 62-69,wherein the first and second plates are rectangular.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f)or 35 U.S.C. § 112(6) is not invoked.

1. A particle sorter comprising: a flow cell; a light source forirradiating an interrogation point of the flow cell; a detector fordetecting light from the interrogation point; a droplet generator forproducing a droplet stream from fluid exiting the flow cell; and adroplet deflector configured to apply a known offset deflection force tothe droplet stream.
 2. The particle sorter according to claim 1, whereinthe droplet deflector comprises first and second plates configured to beoffset from one another.
 3. The particle sorter according to claim 2,wherein the first and second plates are configured to be adjustablyoffset from one another.
 4. The particle sorter according to claim 3,wherein the first and second plates are configured to be adjustablyoffset from one another with respect to a horizontal plane.
 5. Theparticle sorter according to claim 4, wherein the horizontal plane isperpendicular to the axis of the droplet stream.
 6. The particle sorteraccording to claim 4, wherein the first plate comprises an elongatedsection configured to allow the first plate to be adjustably offset fromthe second plate with respect to the horizontal plane.
 7. The particlesorter according to claim 6, wherein the elongated section of the firstplate comprises a set screw configured to allow the first plate to beadjustably offset from the second plate with respect to the horizontalplane. 8-9. (canceled)
 10. The particle sorter according to claim 1,wherein the known offset deflection force is sufficient to offset a dropdeposition position by 2 mm or more.
 11. The particle sorter accordingto claim 1, wherein the known offset deflection force is sufficient tooffset a drop deposition position by one droplet diameter or less. 12.The particle sorter according to claim 1, further comprising a pluralityof partitions configured to receive droplets deflected by the dropletdeflector.
 13. The particle sorter according to claim 12, wherein thepartitions comprise a collection container.
 14. The particle sorteraccording to claim 13, wherein the collection container is a multi-wellplate.
 15. (canceled)
 16. The particle sorter according to claim 12,wherein the partitions comprise collection tubes.
 17. The particlesorter according to claim 11, wherein the diameter of each partition is1.8 mm or less.
 18. The particle sorter according to claim 2, whereinthe first and second plates are configured to be parallel to oneanother.
 19. The particle sorter according to claim 3, wherein the firstand second plates are configured to be adjustably rotated to face oneanother.
 20. The particle sorter according to claim 6, wherein thesecond plate comprises an elongated section configured to allow thesecond plate to be adjustably offset from the first plate with respectto the horizontal plane.
 21. The particle sorter according to claim 20,wherein the elongated section of the second plate comprises a set screwconfigured to allow the second plate to be adjustably offset from thefirst plate with respect to the horizontal plane.
 22. The particlesorter according to claim 3, wherein the droplet deflector furthercomprises an actuator configured to adjust the offset between the firstand second plates.
 23. The particle sorter according to claim 22,wherein the actuator is operably linked to a feedback subsystem. 24-89.(canceled)